Multi-array, multi-specific electrochemiluminescence testing

ABSTRACT

Materials and methods are provided for producing patterned multi-array, multi-specific surfaces for use in diagnostics. The invention provides for electrochemiluminescence methods for detecting or measuring an analyte of interest. It also provides for novel electrodes for ECL assays. Materials and methods are provided for the chemical and/or physical control of conducting domains and reagent deposition for use multiply specific testing procedures.

This application is a continuation-in-part of copending application Ser.No. 08/715,163 filed Sep. 17, 1996 which is a continuation-in-part ofcopending application Ser. No. 08/611,804 filed Mar. 6, 1996, which is acontinuation-in-part of copending applications Ser. Nos. 08/402,076filed Mar. 10, 1995 and 08/402,277 filed Mar. 10, 1995, each of which isincorporated by reference herein in its entirety.

1. INTRODUCTION

The present invention provides for a patterned multi-array,multi-specific surface (PMAMS) for electrochemiluminescence based tests,as well as methods for making and using PMAMS.

2. BACKGROUND OF THE INVENTION 2.1. Diagnostic Assays

There is a strong economic need for rapid sensitive diagnostictechnologies. Diagnostic technologies are important in a wide variety ofeconomic markets including health care, research, agricultural,veterinary, and industrial marketplaces. An improvement in sensitivity,time required, ease of use, robustness, or cost can open entirely newdiagnostic markets where previously no technology could meet the marketneed. Certain diagnostic technologies may possess high sensitivity butare too expensive to meet market needs. Other techniques may be costeffective but not robust enough for various markets. A novel diagnostictechnique which is capable of combining these qualities is a significantadvance and opportunity in the diagnostics business.

There are a number of different analytical techniques used in diagnosticapplications. These techniques include radioactive labeling, enzymelinked immunoassays, chemical calorimetric assays, fluorescencelabeling, chemiluminescent labeling, and electrochemiluminescentlabeling. Each of these techniques has a unique combination ofsensitivity levels, ease of use, robustness, speed and cost which defineand limit their utility in different diagnostic markets. Thesedifferences are in part due to the physical constraints inherent to eachtechnique. Radioactive labeling, for example, is inherently non-robustbecause the label itself decays and the disposal of the resultingradioactive waste results in economic, safety and environmental costsfor many applications.

Many of the sensitive diagnostic techniques in use today aremarket-limited primarily because of the need for skilled technicians toperform the tests. Electrochemiluminescent procedures in use today, forexample, require not only skilled technicians but repeated washing andpreparatory steps. This increases both the costs and the need for wastedisposal. Novel diagnostics which simplify the testing procedures aswell as decrease the cost per test will be of great importance andutility in opening new markets as well as improving performance inexisting markets.

2.2. Electrochemiluminescence

Electrochemiluminescence (“ECL”) is the phenomena whereby anelectrically excited species emits a photon (see, e.g., Leland andPowell, 1990 J. Electrochem. Soc. 137(10):3127-3131). Species from whichECL can be induced are termed ECL labels and are also referred to hereinas TAGs. Commonly used ECL labels include: organometallic compoundswhere the metal is from, for example, the noble metals of group VIII,including Ru-containing and Os-containing organometallic compounds suchas the Ru(2,2′-bipyridine)₃ ²⁺ moiety (also referred to as “Rubpy” orTAG1), disclosed, e.g., by Bard et al. (U.S. Pat. No. 5,238,808). “TAG1”and “Rubpy” also refer to derivatives of Ru(2,2′-bipyridine)₃ ²⁺.Fundamental to ECL-based detection systems is the need for an electricalpotential to excite the ECL label to emit a photon. An electricalpotential waveform is applied across an electrode surface, typically ametal surface, and a counterelectrode (see e.g., U.S. Pat. Nos.5,068,088, 5,093,268, 5,061,445, 5,238,808, 5,147,806, 5,247,243,5,296,191, 5,310,687, 5,221,605). The ECL is promoted to an excitedstate as a result of a series of chemical reactions triggered by theelectrical energy received from the working electrode. A molecule whichpromotes ECL of the TAG is advantageously provided, such as oxalate or,more preferably, tripropylamine (see U.S. Pat. No. 5,310,687).

The excitation of a TAG in an ECL reaction typically involves diffusionof the TAG molecule to the surface of an electrode. Other mechanisms forthe excitation of a TAG molecule by an electrode include the use ofelectrochemical mediators in solution (Haapakka, 1982, Anal Chim. Acta,141:263) and the capture of beads presenting TAG molecules on anelectrode (PCT published applications WO 90/05301 and WO 92/14139).Alternatively, ECL has been observed from TAG that was adsorbed directlyon the surface of working electrodes (US Pat. No. 5,324,457), e.g., bynon-specific adsorption (Xu et al., 1994, Langmuir, 10:2409-2414), byincorporation into L-B films (Zhang et al., 1988, J. Phys. Chem.,92:5566), by incorporation into self-assembled monolayers (Obeng et al.,1991, Langmuir, 7:195), and by incorporation into thick (micrometer)films (Rubenstein et al., 1981, J. Am. Chem. Soc., 102:6641). Similarly,Xu et al. (PCT published application WO 96/06946) have observed ECL fromTAG molecules intercalated into DNA strands when such strands wereadsorbed onto gold electrodes by interaction with aluminum centersimmobilized on a self-assembled monolayer of alkanethiolates.

Various apparatus well known to the art are available for conducting anddetecting ECL reactions. For example, Zhang et al. (U.S. Pat. No.5,324,457) discloses exemplary electrodes for use in electrochemicalcells for conducting ECL. Leventis et al. (U.S. Pat. No. 5,093,268)discloses electrochemical cells for use in conducting ECL reactions.Kamin et al. (U.S. Pat. No. 5,147,806) discloses apparatus forconducting and detecting ECL reactions, including voltage controldevices. Zoski et al. (U.S. Pat. No. 5,061,445) discloses apparatus forconducting and detecting ECL reactions, including electrical potentialwaveform diagrams for eliciting ECL reactions, digital to analogconverters, control apparatus, detection apparatus and methods fordetecting current generated by an ECL reaction at the working electrodeto provide feedback information to the electronic control apparatus.

2.3. Commercial ECL Assays

The light generated by ECL labels can be used as a reporter signal indiagnostic procedures (Bard et al., U.S. Pat. No. 5,221,605). Forinstance, an ECL label can be covalently coupled to a binding agent suchas an antibody or nucleic acid probe. The ECL label/binding agentcomplex can be used to assay for a variety of substances (Bard et al.,U.S. Pat. No. 5,238,808). The use of ECL in assays is reviewed in detailby, for example, Knight et al., 1994, Analyst, 119:879-890. In brief,the ECL technique may be used as a method of detecting in a volume of asample an analyte of interest present in the sample in relatively smallconcentrations.

To date, all commercial ECL assays are carried out on centimeter scaleelectrode surfaces. The centimeter scale electrodes strike a balancebetween the enhanced magnitude of an ECL signal resulting from largerelectrodes and the desirability of decreasing the total sample volumenecessary for each assay. However, even centimeter scale electrodes failto achieve the sensitivity required for many assays. In an attempt toovercome this problem, all commercial ECL systems further enhancesensitivity by using coated magnetic beads to capture ECL analytes orreagents. The beads are then moved adjacent to a working electrode forenhanced sensitivity.

However, the currently available technology has many limitations(primarily cost and complexity) that restrict its use in low cost assaysemploying disposable cartridges as well as its use in high throughputsystems that perform multiple assays concurrently.

Leventis et al. (U.S. Pat. No. 5,093,268) has proposed a method ofassaying more than one different analyte simultaneously by the use ofdifferent ECL labels for each analyte, each emitting photons atdifferent wavelengths for each different analyte in a single assay.However, this technique is limited, for example, by the unavailabilityof a sufficient number of effective ECL labels radiating at differentwavelengths and the need to optimize the chemical conditions for eachECL label. These practical constraints have prevented thecommercialization of such multi-wavelength, multi-analyte ECL detectionsystems.

Commercial methods for conducting ECL assays also require that the assaycell, including the electrodes, must be cleaned by any one of a numberof methods, including the use of dilute acids, dilute bases, detergentsolutions, and so forth as disclosed, for example, by U.S. Pat. No.5,147,806.

2.4. Objects of the Invention

It is therefore an object of the present invention to provide a novel,cost effective electrode and disposable for conducting ECL assays.

It is a further object of the present invention to provide a novel andcost effective system for conducting a plurality of ECL assays, eithersequentially or simultaneously and in a preferred embodiment, providingbuilt-in control standards for improved accuracy.

It is a further object of the present invention to provide a cassettecomprising one or more supports suitable for conducting a plurality ofsimultaneous or sequential ECL assays that is also disposable.

It is a further and related object of this invention to reduce the timeand cost of conducting individual assays for analytes of interest inbiological samples.

It is still a further and related object of this invention to providemethods and apparatus for conducting a plurality of simultaneous assaysfor a plurality of analytes of interest in a single biological sample.

3. SUMMARY OF THE INVENTION

The invention relates to a cassette for conducting ECL reactions andassays comprising one or more binding domains immobilized on a support.The support may act as an electrode for generatingelectrochemiluminescence. Alternatively, one or more electrodes may beon additional supports, and said electrodes may be brought intoproximity to the first support so as to generate ECL. The cassette mayhave one or more electrodes or one or more electrode/counterelectrodepairs. The cassette may also comprise a second support capable of beingplaced adjacent to the first support to provide sample containing meanstherebetween, and/or serve as an electrode. The binding domains arepatterned on a support surface and are prepared so as to bind analytesor reagents of interest.

The invention further relates to novel, disposable electrodes amenableto use in a disposable format. These electrodes can be comprised ofvarious forms of carbon such as glassy carbon, carbon black or carbon(graphitic) nanotubes.

The invention further relates to composite electrodes, i.e. electrodescomprised of more than one material. These electrodes can be tailored tocontrol performance, cost and manufacturability to make them amenable touse in a disposable format.

The invention further relates to assays in which particles are used assolid-phase supports for binding reagents. Said particles are capturedon a porous electrode by filtration and analytes are detected. Kitsbased on pre-prepared conducting filters with particles are described.

The invention further relates to electrodes that can be used to resolvetwo or more ECL signals. Methods for the modification of electrodes arealso described.

The invention further relates to an apparatus for measuringelectrochemiluminescence of a sample that provides support or cassettehandling means, voltage control means adapted to apply a controlledvoltage waveform effective to trigger electrochemiluminescence, photondetector means for detecting electrochemiluminescence from the sampleand sample handling means.

The invention further relates to methods for using the cassettes formeasuring electrochemiluminescence in a sample by contacting theplurality of binding domains of a cassette with a sample which containsa plurality of analytes of interest, under ECL assay conditions, andthen applying a voltage waveform effective to triggerelectrochemiluminescence and detecting or measuring of the triggeredelectrochemiluminescence.

The invention also provides for kits comprising components includingcassettes suitable for simultaneously measuring a plurality ofelectrochemiluminescence reactions, support surfaces and upon which aplurality of domains are immobilized assay, media for conduct of the ECLassay conducting chemical reactions.

The invention is also in rapid disposable electrochemiluminescenceassays. Commercial ECL assays are performed using a flow cell with aworking and counter electrode. A disposable electrode, as disclosedherein, does not require washing and/or cleaning to eliminate carry-overand regenerate a uniform electrode surface as does a permanent flow cellelectrode.

The invention also provides for increased kinetics through the use ofporous electrodes. Formatted and/or porous disposable electrodes areused to rapidly produce assay results. Assay results with disposableelectrodes may be achieved in less than an hour. In preferredembodiments ECL assay results from disposable electrodes may be achievedin less than 30 minutes and in some cases less than 15 minutes. In themost preferred embodiments, the assay results can be achieved in lessthan 5 minutes or in the most advantageous case, than 1 minute. Inmulti-assay formats of the invention more than one ECL assay result maybe achieved in such time periods or less. Kits for rapid disposable ECLsystems are disclosed.

Additionally, the invention provides for portable ECL diagnosticinstruments. Cartridges or kits for portable ECL diagnostics may use thenovel disposable electrodes and reagent packs. PMAMS and electrodes forECL assays may be packaged as kits for use in portable ECL instrumentreaders. Such kits and ECL instrument readers may be used to achieveassay results in short time periods. Assay results may be achieved inthe very short time periods discussed above.

4. DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a cassette according to the invention wherein aplurality of binding domains are present on an electrode.

FIG. 1A illustrates two supports forming a cassette according to theinvention wherein a plurality of binding domains 14 are present onsupport 10 and a plurality of corresponding electrodes 16 is present onsupport 12 so that approximation of the supports places an electrodepair adjacent to each binding domain.

FIG. 2 illustrates two supports forming a cassette according to theinvention wherein a plurality of binding domains 30 on support 26 areadjacent to each of single electrodes 32 so that approximating supports26 and 28 places each of counterelectrodes 38 adjacent to each ofbinding domains 30.

FIG. 3 illustrates two supports forming a cassette according to theinvention wherein a plurality of binding domains 48 have electrodecounterelectrode pairs 50 adjacent thereto on support 44. Support 46 mayoptionally be placed adjacent to support 44 so that support 46 providessample containing means adjacent to binding domains 48 and electrodes50.

FIG. 4 illustrates two supports forming a cassette according to theinvention wherein a plurality of binding domains 64 on support 60 arecontacted with a sample suspected of containing an analyte. Support 62has regions 66 containing reaction medium for detecting or measuring ananalyte of interest or for carrying out a desired reaction so thatapproximating support 60 and support 62 causes binding domains 64 andregions 66 to contact one another.

FIG. 5A illustrates a top view of patterned binding domains for amulti-array, multi-specific binding surface. Geometric shapes,triangles, squares and circles, represent binding domains specific fordifferent analytes. The binding domains may be hydrophobic orhydrophilic. The surrounding surface may have the opposite property(hydrophilic or hydrophobic) of the binding domains to minimizespreading of binding reagents or analyte from the binding domains.

FIG. 5B illustrates a top view of a microfluidics guide for deliveringbinding reagents and/or analytes to discrete binding domains. Each dotillustrates a cross section of a microfluidics guide (e.g., acapillary).

FIG. 5C illustrates a side view of a microfluidics guide showing theapproximation of registered or aligned microfluidic guides fordelivering binding reagents and/or analytes to a multi array ofpatterned binding domains. Each microfluidic guide may deliver adifferent binding reagent to a discrete binding domain.

FIG. 6A illustrates the approximation of a multi-array of electrodes inregister with a surface having patterned multi-array, multi-specificbinding domains. A removable electrode protection barrier is shownbetween the electrode array and the binding surface array. The entireassembly comprises a cassette for conducting a plurality of ECLreactions.

FIG. 6B illustrates the approximation of an array of registered oraligned addressable working and counterelectrodes. The electrodes may beshape complementary with the binding domain or of other shapes (e.g.,interdigitating).

FIG. 7 illustrates the side view of an approximated array of registeredor aligned addressable working and counterelectrodes and thecomplementary binding surface wherein conducting polymers are grown fromthe surfaces of the electrodes across the gap between the electrodearray and the binding domains so as to extend the potential field aroundthe ECL label of the sample to increase the efficiency of the ECLreaction.

FIG. 8 illustrates the side view of an approximated array of registeredor aligned addressable working and counterelectrodes and thecomplementary binding surface with conducting particles interspersedbetween both components to extend the potential field. By extending thepotential field around the ECL label of the sample the efficiency of theECL reaction is enhanced. The conducting particles can be magnetic topermit ready manipulation.

FIG. 9 illustrates the side view of an approximated array of registeredor aligned addressable working and counterelectrodes and thecomplementary binding surface wherein the electrodes have fineprojections extending into the gap between the electrode surface and thebinding domains in order to extend the potential field around the ECLlabel of the sample, to increase the efficiency of the ECL reaction.

FIG. 10 illustrates the side view of an approximated array of registeredor aligned addressable working and counterelectrodes and thecomplementary binding surface where the surfaces are not parallel, butare instead conformed one to the other in a complementary fashion.

FIG. 11 illustrates the side view of a support having a metallic layerthereon to provide a single electrode and binding surface assembly inthe form of a cassette. An array of self-assembled monolayers (“SAMs”)is patterned on the metallic layer.

FIG. 12 illustrates the side view of a support having a metallic layerthereon to provide a single electrode and binding surface assembly inthe form of a cassette. An array of SAMs is patterned on the metalliclayer and conducting microparticles are shown interspersed among thepatterned SAMs so as to extend the potential field around the ECL labelof the sample, to increase the efficiency of the ECL reaction.

FIG. 13 illustrates the side view of a support having a metallic layerthereon to provide a single electrode and binding surface assembly inthe form of a cassette. An array of self assembled monolayers or SAMs ispatterned on the metallic layer and the growth of a conducting polymerand/or fiber from the ECL label so as to extend the potential fieldaround the ECL label of the sample to increase the efficiency of the ECLreaction, is illustrated.

FIG. 14 is a diagram of a support having an array of electrode pairscontrolled by a computer.

FIG. 15 is a diagram of a support having an array of electrode pairs.

FIG. 16 is a diagram of a support having an array of electrode pairs andcomputer system for controlling the energization of each electrode pair.

FIG. 17 is a diagram of a support having an array of electrode pairs anda computer system with a plurality of voltage sources and multiplexersfor controlling the energization of each electrode pair.

FIG. 18 is a diagram of a support having an array of electrode pairs anda computer system with a plurality of switched voltage sources forcontrolling the energization of each electrode pair.

FIGS. 19(a)-(e) are plan views of several alternativeelectrode-counterelectrode pair combinations.

FIG. 20 illustrates a support with a completed sandwich assay.

FIG. 21 illustrates two opposing PMAMS surfaces on supports.

FIG. 22A illustrates an array of microfluidics guides (2201) and afibril mat (2200).

FIG. 22B illustrates binding domains (2202) on a fibril mat (2200).

FIG. 23A illustrates an apparatus for forming a fibril mat by vacuumfiltration.

FIG. 23B illustrates a fibril mat (2304) on a filter membrane (2303).

FIG. 24 illustrates the use of rollers to produce fibril mats.

FIG. 25 shows a schematic of a multi-layer fibril mat, in which theupper layer has binding domains used for assays.

FIG. 26 shows a schematic of a fibril derivatized with moieties thatenhance non-specific binding, and several species, both biological andnon-biological are bound to the surface.

FIG. 27 shows a schematic of a fibril derivatized with moieties thatenhance non-specific binding and several species bound to a derivatizedfibril with some species additionally bound to ligands.

FIG. 28 illustrates several species covalently attached to a fibril andsome species are further bound to additional entities.

FIG. 29 illustrates the use of a multilayer fibril mat as an opticalfilter that, depending on the position of a source of light on or withinthe mat, may allow light to pass and/or may absorb and/or scatter light.

FIG. 30A illustrates cyclic voltammograms from electrochemicalmeasurements on carbon fibril mat electrodes.

FIG. 30B illustrates cyclic voltammograms from electrochemicalmeasurements on gold foil electrodes.

FIG. 31 compares an electrochemical property of fibril mats as afunction of the thickness of the mat and the scan rate.

FIG. 32 shows a plot that illustrates that non-specific binding onfibrils generally increases as the concentration of fibrils in a proteinsolution increases.

FIG. 33 demonstrates that the use of surfactants can reduce non-specificbinding between ECL-TAG1-labeled protein and carbon fibrils.

FIG. 34 shows a schematic of a top view of an experimental cell used tomeasure electrochemical properties and ECL on a fibril mat electrode.

FIG. 35 shows an ECL signal obtained using a fibril mat as an electrodeand 1000 pM TAG1 (solid line) in solution and a signal from assay buffer(no TAG1) (dashed line).

FIG. 36 shows a schematic of a two surface PMAMS device, in which twoarrays of supported electrodes are separated by a patterned dielectriclayer.

FIG. 37 illustrates an apparatus with a plurality of binding domains(3702) on one support and an electrode and counterelectrode on anothersupport.

FIG. 38 shows a cassette where binding domains are presented on thesurfaces of distinct objects supported on the counter electrode.

FIG. 39 shows a gel in contact with a working and counterelectrode.

FIG. 40 shows a graph of ECL intensity and a cyclic voltammogram from anECL labeled gel in contact with a working and counterelectrode.

FIG. 41 shows a graph of ECL intensity and a cyclic voltammogram from anon-ECL labeled gel in contact with a working and counterelectrode.

FIG. 42 shows a schematic for a two-surface cassette used for ECL.

FIG. 43 demonstrates that fibril mats can be used as electrodes for ECLof Antibody-TAG1 adsorbed to the mats.

FIG. 44A shows ECL intensity of a TAG1 labeled protein immobilized on anelectrode.

FIG. 44B shows the cyclic voltammogram of a coated electrode.

FIG. 45A shows quasi-reversible repetitive generation of ECL signal froman immobilized ECL TAG1 labeled protein.

FIG. 45B shows the cyclic voltammogram of a coated electrode indicatingpartial preservation of the coating.

FIG. 46A shows irreversible generation of ECL signal from an immobilizedECL TAG1 labeled protein.

FIG. 46B shows the cyclic voltammogram of a coated electrode indicatingsubstantial loss of the coating.

FIG. 47 shows a multi-array ECL apparatus and a microprocessorcontaining controller means for generating and analyzing ECL signals.

FIG. 48 shows the dose response for an AFP immunoassay that involvesformation of a sandwich complex on streptavidin-coated Dynal beads,capture of the beads on a fibril mat electrode, and detection of thebound complex by ECL.

FIG. 49 shows the dose response for an AFP immunoassay that involves theformation of a sandwich complex on streptavidin-coated silica particles,the capture of the particles on a fibril mat electrode, and detection ofthe bound complex by ECL.

FIG. 50 shows a schematic describing the use of a SAM for immobilizingbinding reagents on a surface.

FIG. 51 shows the dose response for an AFP immunoassay that involves theformation of a sandwich complex on a streptavidin-coated SAM ofalkanethiolates on a gold electrode, and detection of the bound complexby ECL.

FIG. 52 illustrates the presentation of TAG moieties to the workingelectrode in a “Two Surface” assay.

FIG. 53 shows the dose response for an AFP immunoassay that involvesformation of a sandwich complex on a streptavidin-coated, oxidized,EVA-fibril composite and detection of the bound complex by ECL.

FIG. 54 shows the dose response for a nucleic acid hybridization assaythat involves formation of a nucleic acid sandwich complex on astreptavidin-coated, oxidized, EVA-fibril composite and detection of thebound complex by ECL.

FIG. 55 shows the dose response for a DNA assay that involveshybridization of a biotin-labeled oligonucleotide to a TAG1 labeledoligonucleotide, capture of the complex on a streptavidin-coated fibrilmat electrode and detection of the bound complex by ECL.

FIG. 56 shows the dose response for an AFP assay that involves theformation of a sandwich complex on a streptavidin-coated, UTFM on anylon membrane and detection of the bound complex by ECL.

FIG. 57 shows the dose response for an AFP assay that involves theformation of a sandwich complex on a streptavidin-coated UTFM formed ona gold-coated nylon membrane and detection of the bound complex by ECL.

FIG. 58 illustrates an ECL signal in which the electrochemical potentialfor one or more components is shifted.

FIG. 59 illustrates an ECL signal in which the intensity of the ECLsignal for one or more components of the sample is reduced relative tothe ECL signal for other components of the sample.

FIG. 60 shows an ECL trace of a sample that is ECL assay buffer.

FIG. 61 shows an ECL trace of a sample that contains AFP.

FIG. 62 shows a plot of the ECL signal (S-B, the difference between theECL Signal (S) and the background signal (B)) as a function of theconcentration of AFP (IU/mL) for an AFP assay. The ECL mediated AFPassay was conducted using plasma treated fibril-polymer composites as asupport for binding reagents and as a working electrode.

FIG. 63 shows a plot of the ECL signal (S-B, the difference between theECL Signal (S) and the background signal (B)) as a function of theconcentration of AFP (IU/mL) for an AFP assay. The ECL mediated AFPassay was conducted using plasma treated fibril-polymer composites as asupport for binding reagents and as a working electrode.

FIG. 64 shows a plot of the ECL signal (S-B, the difference between theECL Signal (S) and the background signal (B)) as a function of theconcentration of AFP (IU/mL) for an AFP assay. The ECL mediated AFPassay was conducted using plasma treated fibril-polymer composites (15%fibrils by weight) as a support for binding reagents and as a workingelectrode.

FIG. 65 shows a plot of the ECL signal (S-B, the difference between theECL Signal (S) and the background signal (B)) as a function of theconcentration of AFP (IU/mL) for an AFP assay. The ECL mediated AFPassay was conducted using plasma treated fibril-polymer composites as asupport for binding reagents and as a working electrode.

FIG. 66 shows a plot of the ECL signal (S-B, the difference between theECL Signal (S) and the background signal (B)) as a function of theconcentration of AFP (IU/mL) for an AFP assay. The ECL mediated AFPassay was conducted using plasma treated fibril-polymer composites as asupport for binding reagents and as a working electrode.

FIG. 67 shows a plot of the ECL signal (S-B, the difference between theECL Signal (S) and the background signal (B)) as a function of theconcentration of AFP (IU/mL) for an AFP assay. The ECL mediated AFPassay was conducted using dry reagents and without a wash step.

FIG. 68 shows a schematic diagram of an assay cell according to anembodiment of the present invention.

FIG. 69 shows a schematic diagram of an assay system according toanother embodiment of the present invention.

5. DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the invention includes in a broad aspect cassettes forconducting one or more electrochemiluminescence assays. The cassettesare formed of supports having thereon a plurality of binding domainsable to specifically bind one or more analytes of interest. The bindingdomains are prepared as patterned, multi-array multi-specific surfaces(“PMAMS”) on the support. The PMAMS offer a significant improvement fromECL assay methods previously known by, e.g., greatly increasing thedensity of assays that can be performed and allowing for a plurality ofdifferent assays that may be rapidly or simultaneously performed.

The cassette may include a plurality of electrodes able to selectivelytrigger ECL emission of light from ECL labeled reagents bound to thebinding domains. FIG. 47 shows an multi-array ECL apparatus using acassette 4700 which comprises a housing 4717, electrical connections tothe electrode in the cassette 4718, a waveform generator or potentiostat4719, a CCD camera for imaging the ECL emitted from the PMAMS 4720, anda microcomputer for controlling the waveform generator and analyzing theimage received by the camera 4721.

In the embodiment of the invention shown in FIG. 1, a cassette 180comprises a working electrode comprising a conducting material 181 on asupport material 182. A plurality of binding domains, i.e. a PMAMS 183are present on the electrode 181. The cassette also includes a means forintroducing samples and reagents (fluid channel 184) and a counterelectrode 185. A reference electrode 186 may also be included.

In another embodiment, a plurality of working electrodes are used tosimultaneously generate an ECL signal at a plurality of binding domains.In this embodiment, the ECL signal from each binding domain isidentified without the use of light imaging equipment.

In certain embodiments of the invention, it is desirable to reproduciblyimmobilize a specified or predetermined amount of one or more reagentson a surface. Immobilization broadly applies to any method by which areagent is attached to a surface, including but not limited to: covalentchemical bonds; non-specific adsorption; drying a reagent on a surface;electrostatic interactions; hydrophobic and/or hydrophilic interactions;confinement or entrainment in liquids or gels; biospecific binding,(e.g., ligand/receptor interactions or hybridization ofoligonucleotides); metal/ligand bonds; chelation, and/or entanglement inpolymers.

The amount of reagent immobilized on a surface may be predetermined inseveral ways. For example, the amount of reagent on a surface may bespecified by one or more volume and/or area elements in which thereagent is present. It may also be specified by the number of individualmolecules of a reagent that are immobilized on a surface. The amount ofreagent may be specified in terms of the density of a particular reagentin a given region. The amount of reagent may be specified as apercentage of a surface bearing a particular reagent, either with regardto the total area of the surface, or relative to the amounts of otherreagents present on the surface. The amount of reagent may also bedefined as the quantity of reagent that must be present on a particularsurface to give sufficient ECL intensity so as to make an assay achievea desired specificity. In a specific example, a 1 cm² area of a goldsurface may be coated with a monolayer of alkanethiols.

Reagents may also be reproducibly immobilized on coated surfaces. Thecoating may serve to enhance immobilization for some reagents and/orreduce or prohibit immobilization for other reagents. The surface may becompletely coated or the surface may be partially coated (i.e. apatterned coating). The coating may be uniform in composition, or it maycontain elements of different composition. In a specific example, thecoating may be a patterned monolayer film that immobilizesimmunoglobulin G via covalent chemical bonds in some areas, and preventsits immobilization in others.

The coating may also serve to predetermine the amount(s) of one or morereagents immobilized on the surface in subsequent steps or processes.Alternatively, the amount of a particular reagent may be controlled bylimiting the amount of reagent that is deposited.

Having a surface that has reagents (or a coating) immobilized in aquantitative, reproducible fashion gives the ability to reproducibly andquantitatively measure an ECL signal from a sample, thus allowingcalibration.

Broadly, the assays conducted using cassettes according to the inventionare assays that benefit from the use of a plurality of discrete bindingdomains. For example, use of such cassettes allows rapid and/orconcurrent detection or measurement of a wide variety of analytes ofinterest. In a preferred embodiment, the assays according to theinvention are also those that benefit from the use of an ECL labeledreagent, analyte or binding surface. An ECL assay according to theinvention comprises contacting a plurality of binding domains with asample suspected of containing an analyte of interest and triggering anECL emission from a bound ECL label, wherein the ECL label is on theanalyte or a competitor of the analyte, on a reagent that binds to theanalyte or on the plurality of binding domains.

The invention provides for ECL assay methods for detecting or measuringan analyte of interest, comprising (a) contacting one or more bindingdomains immobilized on an electrode, in which said contacting is with asample comprising molecules leveled to an ECL label, (b) applying avoltage waveform effective to trigger ECL at said binding domains, and(c) measuring or detecting ECL.

The term sample is used in the broadest sense. It includes a quantity ofany substance to be used in the methods of the invention. By way ofnon-limiting examples it may include a portion of a material to beassayed containing an analyte-of-interest, a pre-processed or preparedpart thereof or a quantity of reagents to be used in the method of theinvention.

The invention also provides ECL assay methods for detecting or measuringan analyte of interest, comprising (a) contacting one or more bindingdomains, said binding domains being immobilized on a surface of one ormore supports, in which said contacting is with a sample comprisingmolecules linked to an electrochemiluminescent label; (b) bringing anelectrode into proximity to said binding domains; and (c) applying avoltage waveform effective to trigger ECL at said binding domains; anddetecting or measuring ECL.

In another embodiment, the invention provides ECL assay methods for (a)contacting one or more binding domains, said plurality of bindingdomains (i) being immobilized on a surface of one or more supports, and(ii) being spatially aligned with and in proximity to a plurality ofelectrode and counterelectrode pairs, in which said contacting is with asample comprising molecules linked to an electrochemiluminescent label;(b) bringing an electrode and counterelectrode into proximity to saidbinding domains; (c) applying a voltage waveform effective to triggerelectrochemiluminescence at said binding domains; and (d) detecting ormeasuring electrochemiluminescence.

The invention provides a method of detecting in a volume of amulticomponent, liquid sample a plurality of analytes of interest whichmay be present in the sample at various concentrations.

Broadly a plurality of analytes may be detected from a multicomponentsample in less than 10⁻³ molar concentrations. Preferably a plurality ofanalytes may be detected at less than 10⁻¹² molar concentrations from amulticomponent sample.

The invention provides for detection from a multicomponent sample whichmay be performed as heterogeneous assays, i.e., assays in which aplurality of unbound labeled reagents are separated from a plurality ofbound labeled reagents prior to exposure of the bound labeled reagentsto electrochemical energy, and homogeneous assays, i.e., assays in whicha plurality of unbound labeled reagents and bound labeled reagents areexposed to electrochemical energy together.

In the assays of the present invention, the electromagnetic radiationused to detect a particular analyte is distinguishable from theelectromagnetic radiation corresponding to other analytes by identifyingits position and/or location as one or more features of a pattern, saidpattern corresponding to the pattern of the binding domains in thePMAMS.

In the homogeneous assays of the present invention, the electromagneticradiation emitted by the bound labeled reagents either as an increase oras a decrease in the amount of electromagnetic radiation emitted by thebound labeled reagents in comparison to the unbound reagents, or bydetection of electromagnetic radiation emitted from sourcescorresponding in space to one or more features of a patterncorresponding to the pattern of the binding domains in the PMAMS.

In a specific example of the method of the invention shown in FIG. 20, asandwich assay is conducted on a support (5) with a plurality of bindingdomains (BD) on its surface that are specific for binding a particularanalyte (An). When a sample suspected of containing the analyte isapplied to the binding domains, the analyte is bound to the bindingdomains. Antibodies (Ab), which are suitable for selectively bindinganalyte (An) and have been labeled with an ECL moiety (TAG) to formAb-TAG, are then applied to the analyte on the binding domains. Afterexcess, unbound Ab-TAG is washed off the binding domains, a potentialwaveform suitable for triggering electrochemiluminescence is applied tothe TAG by electrodes (not shown) to trigger an ECL emission from anyTAG on the binding domains. The ECL signal is detected by lightdetection means and recorded by digital computer means.

Further embodiments, features and variations of the invention areprovided as described hereinbelow.

5.1. Preparation of a Binding Surface

To better understand the invention, a more detailed description of thepreparation of binding domains on a support is provided. A patternedarray of binding domains on a surface that are specific for a pluralityof analytes is referred to herein as a patterned, multi-arraymulti-specific surface or PMAMS. PMAMS are prepared on a support, forexample, by patterning of self-assembled monolayers (“SAMs”) (Fergusonet al, 1993, Macromolecules 26(22):5870-5875; Prime et al., 1991,Science 252:1164-1167; Laibinis et al., 1989, Science 245:845-847; Kumaret al., 1984, Langmuir 10(5):1498-1511; Bain et al., 1989, Angew. Chem.101:522-528). Surface patterning methods also include the use ofphysical etching (e.g., micromachining) (Abbott et al., 1992, Science257:1380-1382; Abbott, 1994, Chem. Mater. 6(5):596-602),microlithography (Laibinis et al., 1989, Science 245:845-847),attachment of chemical groups to the surface through the use ofphotoactivatable chemistries (Sundberg et al., 1995, J. Am. Chem. Soc.117(49):12050-12057), and micro-stamping techniques (Kumar et al., 1994,Langmuir 10(5):1498-1511; Kumar et al., 1993, Appl. Phys. Lett.63(14):2002-2004). Other surface patterning methods include proceduresfor the spatially controlled dispensing of fluids or particles (e.g.,micropen deposition (e.g., using a microfluidic guide to deliver onto asurface using X-Y translation)), microcapillary filling (Kim et al.,1995, Nature 376:581), Ink-Jet technology, or syringe dispensers.Combinations of these techniques may be used to provide complex surfacepatterns. In FIG. 5A, a support 600 is shown with shape independentbinding domains that are represented, simply for illustration purposes,as geometric shapes 602 to indicate that different binding specificitiesmay be present on a single support. Surface 604 between binding domainsmay be alternatively hydrophobic or hydrophilic to confine deposition ofbinding reagent to form binding domains. Binding domains and/or thesurface(s) between binding domains may be alternatively prone andresistant to nonspecific binding, and/or they may be prone and resistantto the attachment of binding reagents via covalent or non-covalentinteractions. In the case where non-specific binding through hydrophobicinteractions is not the desired method for attachment of bindingchemistries to the surface, detergent may be added to prevent incidentalnon-specific binding from occurring.

The binding domains are broadly from 0.1 μm to 10 mm in width ordiameter or widest dimension depending upon the geometry of the domain.The surfaces are selectively derivatized to have specific bindingcomponents exposed to e.g., the ECL assay solution. Additionally,non-specific interactions at the binding domains are decreased whilemaintaining a specific binding moiety by incorporating moieties such aspolyethyleneglycols on the exposed surface of the discrete bindingdomains (Prime et al., 1993, J. Chem Soc. 115:10714-10721; Prime et al.,1991 Science 252:1164-1167; Pale-Grosdemange et al., 1991, J. Am. Chem.Soc. 113:12-20).

The PMAMS may contain broadly from 2 to 10⁸ binding domains. Preferably,the number of binding domains is from 50 to 500. In still otherembodiments, the number of binding domains is from 25 to 100. In stillother embodiments, the number of binding domain is from 2 to 20.

The support may be a variety of materials including but not limited toglass, plastic, ceramic, polymeric materials, elastomeric materials,metals, alloys, composite foils, semiconductors, insulators, siliconand/or layered materials, etc. Derivatized elastomeric supports can beprepared, e.g., as described by Ferguson et al., 1993, Macromolecules26:5870-5875; Ferguson et al., 1991, Science 253:776-778; Chaudhury etal., 1992, Science 255:1230-1232.

The surface of the support on which PMAMS are prepared may containvarious materials, e.g., meshes, felts, fibrous materials, gels, solids(e.g., formed of metals) elastomers, etc. The support surface may have avariety of structural, chemical and/or optical properties. For example,the surface may be rigid or flexible, flat or deformed, transparent,translucent, partially or fully reflective or opaque and may havecomposite properties, regions with different properties, and may be acomposite of more than one material. The surface may have patternedsurface binding regions and/or patterned regions where catalyses mayoccur according to the invention on one or more surfaces, and/or anaddressable array of electrodes on one or more surfaces. The surfaces ofthe supports may be configured in any suitable shapes including planar,spheroidal, cuboidal, and cylindrical. In a specific embodiment, thesupport bearing a PMAMS is a dipstick.

The support bearing a PMAMS may contain carbon, e.g., particulatecarbon, graphite, glassy carbon, carbon black, or may contain one ormore carbon fibers. These fibers may be amorphous or graphitic carbon.

A support bearing a PMAMS may contain “carbon fibrils”, “carbonnanotubes”, “graphitic nanotubes”, “graphitic fibrils”, “carbontubules”, “fibrils” and “buckeytubes”, all of which terms are used todescribe a broad class of carbon materials (see Dresselhaus, M. S.;Dresselhaus, G.; Eklund, P. C.; “Science of Fullerenes and CarbonNanotubes”, Academic Press, San Diego, Calif., 1996, and referencescited therein). We use the terms “fibrils” and “carbon fibrils”throughout this application to include this broad class of carbon-basedmaterials.

Individual carbon fibrils as disclosed in U.S. Pat. Nos. 4,663,230,5,165,909, and 5,171, 560 are particularly advantageous. They may havediameters that range from about 3.5 nm to 70 nm, and length greater than10² times the diameter, an outer region of multiple essentiallycontinuous layers of ordered carbon atoms and a distinct inner coreregion. Simply for illustrative purposes, a typical diameter for acarbon fibril may be approximately between about 7 and 25 nm, and atypical range of lengths may be 1 μm to 10 μm. Carbon fibrils may alsohave a single layer of carbon atoms.

Carbon materials can be made to form aggregates. For example, asdisclosed in U.S. Pat. No. 5,110,693 and references therein, two or moreindividual carbon fibrils may form microscopic aggregates of entangledfibrils. These aggregates can have dimensions ranging from 5 nm toseveral cm. Simply for illustrative purposes, one type of microscopicaggregate (“cotton candy or CC”) resembles a spindle or rod of entangledfibers with a diameter that may range from 5 nm to 20 μm with a lengththat may range from 0.1 μm to 1000 μm. Again for illustrative purposes,another type of microscopic aggregate of fibrils (“birds nest, or BN”)can be roughly spherical with a diameter that may range from 0.1 μm to1000 μm. Larger aggregates of each type (CC and/or BN) or mixtures ofeach can be formed (vide infra).

Fibrils that can be used in a support include but are not limited toindividual fibrils, aggregates of one or more fibrils, suspensions ofone or more fibrils, dispersions of fibrils, mixtures of fibrils withother materials (e.g., oils, paraffins, waxes, polymers, gels, plastics,adhesives, epoxies, teflon, metals, organic liquids, organic solids,inorganic solid, acids, bases, ceramics, glasses, rubbers, elastomers,biological molecules and media, etc.) as well as combinations thereof.

The fibrils may be magnetic in some cases and non-magnetic in others.The extent to which fibrils can be made magnetic or non-magnetic iscontrolled by the process used to produce the fibrils. Examples of suchprocess are disclosed in U.S. Pat. Nos. 4,663,230, 5,165,909, and5,171,560. PMAMS are located on, in, or in proximity to the supportsdescribed supra.

PMAMS can be generated from different types of surface binding groups.Self-assembling monolayers that can be used to form a monolayer on asurface to which they bind, include but are not limited to alkane thiols(which bind gold and other metals), alkyltrichlorosilane (e.g., whichbind silicon/silicon dioxide), alkane carboxylic acids (e.g., which bindaluminum oxides) as well as combinations thereof. The monolayer may beformed first and then linking chemistry used to attach binding reagents.Derivatization after self-assembly produces a more perfecttwo-dimensional crystalline packing of the monolayer on a supportsurface with fewer pin holes or defects. The monolayer can bederivatized with the binding reagents before or after self-assembly.Regular defects in the monolayer may be desirable, and can be obtainedby derivatization prior to self-assembly of the monolayer or the supportsurface. If the derivatized group (e.g., exposed binding group) on thebinding reagent is sterically large, it may create a close-packedsurface at the exposed end, but with regular gaps at the metal surface.This is useful for allowing charge to flow through these regular gaps tothe ECL labeled moieties bound to the portion contacting the samplesolution.

The preparation of incomplete monolayers is known in the art. Otherprocedures for the preparation of incomplete monolayers include but arenot limited to: the formation of monolayers from dilute solutions ofbinding reagent, the termination of the monolayer forming reactionbefore completion, the damaging of more complete monolayers withradiation (e.g., ionic particles), light or chemical reagents. In oneembodiment, repeated stamping without re-inking the stamp can give arange of defective monolayers (Wilbur et al., 1995, Langmuir, 11:825)PMAMS can be generated on the surface of matrices. Matrices may behighly conducting, e.g., metal electrodes or conducting polymer films;or matrices may be insulators; or semi-conducting and/or of mediumconductivity. The matrix material may be an ionic conductor or a porousmaterial. Such porous materials may be utilized as support materialand/or a conductive material and/or a filter material and/or achannelling material (e.g., allowing passage of fluids, ionic speciesetc.).

The porous material may be combined with additional materials. Forexample, composite structures may be fabricated of porous materials withadditional porous materials, conductive materials, semiconductivematerials, channelling structures and/or solutions (e.g., ionic fluids).Such composites may be laminar structures, sandwich structures, and/orinterspersed composites. A solid matrix may be used which is a porousmaterial supported on a metal electrode. Alternatively, a porousmaterial is sandwiched between conducting materials, semiconductingmaterials or a combination of semiconducting and conducting materials.One or more binding domains may be contained on one continuous slab ofthe porous material and/or may be located on a plurality of discreteobjects on the support each with one or more binding domains. The porousmaterial (e.g., gel) surface may be flat, hemispherical or take on anyregular or irregular shape and/or may have a variety of physicalproperties (e.g., elastomeric, rigid, low density, high density,gradient of densities, dry, wet etc.) and/or optical properties (e.g.,transparent, translucent, opaque, reflective, refractive etc.) and orelectrical properties (e.g. conductive, semiconductive, insulating,variably conductive, for example wet vs. dry etc.). The porous materialmay be a composite of more than one materials.

A pattern of channels may be formed in the matrix. The porous materiallayers may be from 5 microns to 2000 microns thick. The porous materiallayers may also be thicker than 2 mm.

The pores may extend partially and/or fully through the material or maybe part of a network of pores. These pores may have dimensions rangingbroadly from 50 Å to 10000 μm. In a preferred embodiment, the materialhas some pores with dimensions ranging from 200 Å to 500 Å and somepores with dimensions ranging from 0.5 μm to 100 μm.

The porosity of the material may be constant throughout the material ormay increase or decrease as a function of the position in the material.The material may have a wide variety of pores of different sizedistributed in a disorganized and/or random manner.

For example, the material may have some pores that are large enough topass objects as large as biological cells, some pores that can passbiological media as large as proteins or antibodies, some pores that canpass only small (<1000 molecular weight) organic molecules, and/orcombinations thereof.

The porosity of the material may be such that one or more molecules,liquids, solids, emulsions, suspensions, gases, gels and/or dispersionscan diffuse into, within and/or through the material. The porosity ofthe material is such that biological media can diffuse (actively orpassively) or be forced by some means into, within and/or through thematerial. Examples of biological media include but are not limited towhole blood, fractionated blood, plasma, serum, urine, solutions ofproteins, antibodies or fragments thereof, cells, subcellular particles,viruses, nucleic acids, antigens, lipoproteins, liposaccharides, lipids,glycoproteins, carbohydrates, peptides, hormones or pharmacologicalagents. The porous material may have one or more layers of differentporosity such that biological media may pass through one or more layers,but not through other layers.

The porous material may be able to support a current due to the flow ofionic species. In a further refinement, the porous material is a porouswater-swollen gel, for example polyacrylamide or agar. A variety ofother gel compositions are available (for example see Soane, D. S.Polymer Applications for Biotechnology; Soane, D. S., Ed.; Simon &Schuster: Englewood Cliffs, N.J., 1992 or Hydrogels in Medicine andPharmacy, Vol. I-III; Peppas, N. A. Ed.; CRC Press: Boca Raton, Fla.,1987). Binding domains can be attached to matrices by covalent andnon-covalent linkages. (Many reviews and books on this subject have beenwritten; some examples are Tampion J. and Tampion M. D. ImmobilizedCells: Principles and Applications Cambridge University Press: NY, 1987;Solid Phase Biochemistry: Analytical and Synthetic Aspects Scouten, W.H. Ed., John Wiley and Sons: NY, 1983; Methods in Enzymology,Immobilized Enzymes and Cells, Pt. B Mosbach, K. Ed., Elsevier AppliedScience: London, 1988; Methods in Enzymology, Immobilized Enzymes andCells, Pt. C Mosbach, K. Ed., Elsevier Applied Science: London, 1987;Methods in Enzymology, Immobilized Enzymes and Cells, Pt. C Mosbach, K.Ed., Elsevier Applied Science: London, 1987; see also Hydrogels inMedicine and Pharmacy, supra). For example, a protein can be attached toa cross linked copolymer of polyacrylamide and N-acryloylsuccinimide bytreatment with a solution of the protein. The binding domains may alsobe integrated into a porous matrix in a step prior to polymerization orgelation. In one embodiment, binding domains may be attached touncrosslinked polymers by using a variety of coupling chemistries. Thepolymers may then be crosslinked (for example using chemistries whichinclude amide bonds, disulfides, nucleophilic attack on epoxides, etc.)(see for example: Pollack et al., 1980, J. Am. Chem. Soc.102(20):6324-36). Binding domains may be attached to monomeric specieswhich are then incorporated into a polymer chain during polymerization(see Adalsteinsson, O., 1979, J. Mol. Catal. 6(3): 199-225). In yetanother embodiment, binding domains may be incorporated into gels bytrapping of the binding domains in pores during polymerization/gelationor by permeation of the binding domains into the porous matrix and/orfilm. Additionally, binding domains may be adsorbed onto the surface ofporous matrices (e.g., polymer gels and films) by nonspecific adsorptioncaused for example by hydrophobic and/or ionic interactions. Biotin maybe advantageously used as a linking or binding agent. Avidin,streptavidin or other biotin binding agents may be incorporated intobinding domains.

PMAMS can be generated on porous materials (e.g., gels) with varyingpore size and solvent content. For example, polyacrylamide gels varyingin pore size can be made by varying the concentration of acrylamide andthe degree of crosslinking.

On such PMAMS with pore sizes smaller than the analyte, bindingreactions will occur substantially on the surface of the gel. In thiscase, filtration and/or electrophoresis through the gel can be used toconcentrate analytes at the surface of the gel and modulate the kinetics(e.g., increase the rate) of the binding reaction. Faster kinetics isadvantageous in rapid assays (e.g., short times to results) and maygenerate increased sensitivity in a shorter time period.

On PMAMS with pore sizes larger than the analyte, binding reactions canoccur on the surface as well as in the bulk of the gel. In this case,filtration can be used and/or electrophoresis can be used to increasethe kinetics of binding and remove unbound species from the surface.

PMAMS formed on gels can be stored wet and/or they may be stored in adried state and reconstituted during the assay. The reagents necessaryfor ECL assays can be incorporated in the gel before storage (bypermeation into the gel or by incorporation during formation of the gel)and/or they can be added during the assay.

Patterned binding domains of a PMAMS can be generated by application ofdrops or microdrops containing each binding domain in the matrix in aliquid form to a substrate. Solidification and/or gelling of the liquidcan then be caused by a variety of well known techniques(polymerization, crosslinking, cooling below the gelling transition,heat). Agents that cause solidification or gelation may be included inthe drops, so that at some time after dispensing, the drops solidifyand/or gel. A subsequent treatment (e.g., exposure to light, radiationand/or redox potential) may be used to cause solidification and/orgelation. In other embodiments such drops or microdrops may be slurries,pre-polymeric mixtures, particulate groups, and/or substantially soliddrops. Additionally vapor phase deposition may be utilized.

Patterning can also be achieved by forming a layered structure ofmatrices each containing one or more binding domains. For example,agarose linked (by standard chemistries) to an antibody could be pouredinto a container and allowed to gel by cooling. Subsequent layerscontaining other antibodies could then be subsequently poured on thefirst layer and allowed to gel. The cross section of this layeredstructure gives a continuous surface presenting a plurality of distinctbinding domains. Such cross sections may be stacked and another crosssection may be cut to create a PMAMS surface with even greater densityof binding domains. Alternatively, lines of a matrix containing a givenbinding element are laid down adjacent to one another and/or stacked.Such structures may also be cut in cross-section and utilized as a PMAMSsurface.

Patterning can also be achieved by taking advantage of the ability ofsome matrices to achieve separation. For example, a mixture of nucleicacid probes could be separated by electrophoresis in a polyacrylamideslab generating a surface presenting a plurality of distinct bindingdomains.

Microfluidics guides may also be used to prepare the PMAMS bindingdomains on a support. A partial list of microfluidic guides includeshollow capillaries, capillaries made of and/or filled with a matrix(e.g., a porous or solvent swollen medium), solid supports which cansupport a thin film or drop of liquid. The capillary may be solid andreagents flow along the outside surface of the capillary, a reagentfluid reservoir may be exposed to a porous matrix tip which is broughtinto contact with a PMAMS surface. For example, the reagent reservoirmay be continuously or periodically refilled so that a given porousmatrix tip may reproducibly deposit reagents (e.g., alkane thiols toform monolayers and/or binding reagents etc.) a plurality of times.Additionally, varying the porosity of the tip can be utilized to controlreagent flow to the surface. Different or identical binding reagents maybe present in a plurality of capillaries and/or multiple distinctbinding agents may be present in a given capillary. The capillaries arebrought into contact with the PMAMS (e.g., patterned SAM) surface sothat certain regions are exposed to the binding reagents so as to creatediscrete binding domains. Different binding reagents, each present in adifferent microfluidic guide are delivered concurrently from the fluidicguide array onto a metal surface, SAM, etc, as desired. Microfluidicguides can also be used to ink a microstamp with a desired moleculeprior to application to the support surface. For example, individualmicrofluidic guides can be used to apply different binding reagentslinked to a moiety that promotes adsorption to the surface of thesupport (e.g., a free thiol on a hydrocarbon linker, which promotesadsorption to gold), to form a PMAMS. Thus, for example, a microstampinked via the use of microfluidic guides with antibodies of differentspecificities that have incorporated a linker with a free thiol, can beused to apply such antibodies in desired areas on a gold surface to formdiscrete binding domains of a PMAMS.

Microfluidic guide also refers to microprinting devices which delivermicrodrops of fluid by ejection of the drop through a small orifice(e.g., an Ink-Jet printer). The ejection drops in these devices may becaused by different mechanisms including heating, electrostatic charge,and/or pressure from a piezo device. Patterning of more than one liquidcan be achieved through the use of multiple orifices and/or one orificeand appropriate valving.

In one method for preparation of a PMAMS, microfluidic guides are usedto deliver (preferably concurrently) directly onto discrete regions on asurface, drops containing the desired binding reagents, to form discretebinding domains. The binding reagents may contain a functional chemicalgroup that forms a bond with a chemical group on the surface to which itis applied. In another variation, binding reagents in the drop arenonspecifically adsorbed or bound to the surface (e.g., dried on thesurface).

Alternatively, drop(s) deposited on a surface contain reagents that canform a matrix. This matrix may be a solid, polymer or a gel. Theformation of the matrix may be by evaporation of solvent. It may be bypolymerization of monomeric species. It may be by cross-linking ofpreformed polymers. It may be by modulating temperature (e.g., coolingand/or heating). It may be by other methods. For example, a polymericspecies may be cooled through a cooling transition or by addition of areagent that causes gelling. The formation of the solid matrix may beinduced by generation of reactive species at an electrode (including thesubstrate), by light (or other radiation) by addition of reagents thatinduce solidification or gelling, by cooling or heating. Additionally,the surface may contain catalysts capable of initiating matrix formation(e.g. gelling or polymerization).

In a preferred technique, patterned hydrophilic/ hydrophobic regions toprevent spreading of applied fluids or gels can be used. Such a fluid orgel may contain binding reagents to be linked to a surface on a supportto form a binding domain of the PMAMS. In this case, use of such ahydrophilic/hydrophobic border aids in confining the produced bindingdomain to a discrete area. Alternatively, the fluid contains reagentswhich can form a matrix on the surface and binding reagents arecontained within a defined region when deposited on a surface. Forexample, hydrophilic/hydrophobic border aids may be utilized to confinethe drop to a defined region. Additionally, either the hydrophilic orhydrophobic areas may present groups which can be incorporated (e.g.,covalently or non-covalently bound) into the matrix, allowing for a morestable adhesion of the matrix to the substrate (Itaya and Bard, 1978,Anal. Chem. 50(11):1487-1489). In another technique, the fluid or gelthat is applied is the sample containing the analyte of interest, andthe sample is applied to a prepared PMAMS. In one preferred example,capillaries containing hydrophilic solutions can be used to deposit asolution onto discrete areas, creating hydrophilic domains surrounded byhydrophobic regions. Alternatively, hydrophobic binding domainssurrounded by hydrophilic regions can be used with a hydrophobic fluidcontaining binding reagents or analyte(s)). Hydrophobic and hydrophilicare relative terms, with respect to each other and/or with respect tothe sample to be applied, i.e., such that the spread or wetting of afluid or gel sample applied to the binding domains is controlled.Further, controlled solution deposition from the microfluidics array maybe accomplished using physical surface features (e.g., wells or channelson the surface). A microfluidics guide can be included in a cassette, ormore preferably, used to apply specific reagents to a support prior touse.

More than one linking chemistry may be applied to the same supportsurface and/or a surface with both hydrophilic and hydrophobic bindingdomains can be created using multiple stamps. For example, an area wherea hydrophilic binding domain is desired at position 1 and a hydrophobicbinding domain is desired at position 2 can be prepared as follows. Afirst hydrophilic stamp is made which has a disk at position 1 and alarger ring at position 2. A second hydrophobic stamp is made with adisk at position 2 which fits inside the ring monolayer left by stamp 1.Finally, the surface is washed with a hydrophobic solution of monolayercomponents.

In particular, a PMAMS is generated by micro-contact printing, i.e.,stamping. The monolayer so applied is composed of a surface-bindinggroup, e.g., for a gold surface, a thiol group with an alkane (e.g.,(CH₂)_(n))) spacer is preferred. A spacer group is linked (preferablycovalently bound) to a linking group A. “A” can be, e.g., avidin,streptavidin or biotin or any other suitable binding reagent with anavailable complementary binding partner “B”. The A:B linkage may becovalent or non-covalent and some linkage chemistries known to the artthat can be used are disclosed by, e.g., Bard et al. (U.S. Pat. Nos.5,221,605 and 5,310,687). “B” is further linked to a binding reagentsuch as an antibody, antigen, nucleic acid, pharmaceutical or othersuitable substance for forming a binding domain that can bind to one ormore analytes of interest in a sample to be tested. B may also be linkedto an ECL TAG or label. Linking group B may be delivered to the SAM bymeans of a capillary or microfluidics guide array (FIGS. 5A-5C) able toplace a plurality of “B” reagents with different binding surfacespecificities on the monolayer “A” linkage. A and B can also be linkedbefore or prior to being attached to the monolayer. As discussed, inFIG. 5A, shape independent binding domains are represented, simply forillustration purposes as geometric shapes 602 to indicate that differentbinding specificities may be present on a single support 600. FIG. 5Bprovides a top view of a microfluidic guide (e.g., capillary) array 606.The dots 610 are the guides in cross section. FIG. 5C provides a sideview of a microfluidic guide array 608. The lines emerging from the topand bottom are individual microfluidic guides 610. The geometric shapes612 on the lower aspect represent specific binding domains formed upondelivery of binding reagent from each individual capillary.

By way of example, after the first stamping discussed supra, the baresurface (e.g., gold) regions may be reacted with a second alkane thiolwhich does not have linking chemistry A and is of the oppositehydrophobicity/hydrophilicity of the first monolayer above. In this way,specific linking domains are prepared on a surface.

A binding reagent that is specific or for one analyte of interest may beused for each binding domain or a binding reagent may be used thatspecifically binds to multiple analytes of interest.

In yet another variation, a support surface may be stamped multipletimes by materials (e.g., binding reagents, ECL labels, SAMs) havingdifferent linking chemistries and/or binding moieties as shown by FIG.5A above.

The binding reagents that are patterned can be stable and/or robustchemical groups (e.g., that survive the conditions to which they aresubjected) which are later linked to less stable or robust bindinggroups. Multiple binding linkages may be utilized so as to optimize theconditions of each step in the preparation of a PMAMS surface and/orsimplify the-manufacturing of PMAMS surfaces. For example, a first PMAMSsurface may be fabricated in a generic fashion and then modified tocreate different PMAMS surfaces. In another example, a generic PMAMSsurface may be reacted with a solution mixture of binding reagents whichthemselves contain binding domains which direct them to particularregions (e.g., binding domains) on the PMAMS surface. For example, apattern of binding domains each presenting a different oligo(nucleotide)sequence is linked to the surface. This surface is then treated with asolution containing a mixture of secondary binding reagents, each linkedto a oligo (nucleotide) sequence complementary to a sequence on thesurface. In this way, patterning of these secondary binding elements canbe achieved. Preferably, the oligo (nucleotide) sequences are 6-30 mersof DNA. Certain sets of 6-30 mer sequences may contain substantiallysimilar sequence complementarity so that the approximate bindingconstants for hybridization are similar within a given set anddiscernably different from less complementary sequences. In anotherembodiment, the secondary binding elements are proteins (for example,antibodies).

Methods described to inhibit wetting or spread of applied reagents orsample on a surface as described in Section 5.13 infra, can also be usedin the preparation of PMAMS (and/or in sample application). Appliedpotential (e.g., from the electrode/counterelectrode pair) may be usedto further control the deposition and/or spreading of reagents and/orsamples (see, e.g., Abbott et al., 1994, Langmuir 10(5):1493-1497).

The PMAMS binding reagents may be located on materials that containcarbon, e.g. particulate carbon, carbon black, carbon felts, glassycarbon and/or graphitic carbon. In some embodiments, they may be locatedon carbon fibers, e.g. carbon fiber, or carbon fibrils. The bindingreagents may be located on individual carbon fibrils or they may belocated on aggregates of one or more fibrils. In many embodiments, thePMAMS binding reagents may be located on suspensions or dispersion ofthese carbon materials, mixtures of carbon materials with othermaterials as well as combinations thereof.

The PMAMS binding reagents may be located on a plurality of individualfibrils and/or aggregates of fibrils localized on or in or in proximityto a support. In one example, the binding reagents are localized ondispersed individual fibrils or fibril aggregates. These fibrils oraggregates of fibrils may be localized spatially into distinct domainson a support, and may constitute binding domains. In another example,the binding reagents may be located on aggregates of carbon particles.

In another example, individual such binding domains or a plurality ofsuch binding domains are located in spatially distinct regions of thesupport. By way of a non-limiting example, individual such bindingdomains or collections of binding domains may be located in depressions,pits and/or holes in the support. In still another example, individualbinding domains or a plurality of domains may be located in drops ofwater, gels, elastomers, plastics, oils, etc. that are localized on thesurface of the support. In yet another example, individual bindingdomains may be localized on the support by a coating (which may bepatterned) that has different binding affinities for different bindingreagents and/or binding reagent/fibril ensembles.

Binding domains located on a plurality of individual fibrils and/oraggregates of fibrils may be prepared on a support by means of one ormore microfluidic guides (e.g., a capillary). Different or identicalbinding reagents may be present in or on a plurality of microfluidicguides and/or multiple distinct binding agents may be present in or on agiven microfluidic guide. The capillaries may be brought into contactwith the support (spotting) and/or may deliver the reagents while eitherthe microfluidic guide and/or the surface is being scanned or translatedrelative to the other (i.e., a penlike method of writing). Themicrofluidic guide may deliver the binding reagents located on thefibrils to the support so that certain regions of the support areexposed to the fibril-binding reagent complex(es) so as to create adiscrete binding domain(s). In a preferred aspect, different bindingreagents, each present in a different microfluidic guide are deliveredconcurrently from the guide array onto the support. In one example,binding reagents and/or the fibrils on which they are localized arederivatized with a chemical functional group that forms a bond (e.g.,covalent or non-covalent interaction) to the surface of the support. Insome embodiments, the binding reagents and fibrils are non-specificallybound or adsorbed to the surface. In yet another aspect, the bindingreagents localized on the fibrils may be delivered to depressions, pitsand/or holes in the surface of the support. In another example, thebinding reagents are delivered to a surface that is coated with amaterial that has a stronger or weaker binding affinity for certainbinding reagents or binding reagent/fibril ensembles and so createsdomains of the reagents that are localized spatially and distinctly fromother binding reagents

The binding reagents are localized on one or more individual fibrils oraggregates of fibrils that are magnetic. In such a case, a magneticsupport may attract the binding reagents localized on magnetic fibrilsto the support.

The support may contain several distinct regions that are magnetic andare surrounded by regions that are not magnetic. Binding reagentslocalized on magnetic fibrils may be localized on magnetic regions ofthe support. In one example, the support may contain one or moredistinct regions that are magnetic and are surrounded by regions thatare not magnetic, and the strength of the magnetic field in the magneticregions can be modulated or switched. In this aspect, use of such amodulated or switchable magnetic field aids in affixing or releasing thebinding reagents localized on fibrils from the surface of the supportand so may serve to stir or mix said domains.

There are broadly from 2 to 10⁸ binding domains and preferably from 25to 500 domains.

The binding domains may be located on the working electrode and/or thecounter electrode.

The different embodiments described herein for different types of PMAMS,supports, and electrodes and configurations thereof may also bepracticed in combination with each other.

The PMAMS supports may be preserved (e.g., through protective surfacecoatings, drying the surface, robust packaging under vacuum or inertatmosphere, refrigeration and related methods) for later use.

5.2. Binding Reagents

The binding domains of the invention are prepared so as to containbinding reagents that specifically bind to at least one analyte (ligand)of interest. Binding reagents in discrete binding domains are selectedso that the binding domains have the desired binding specificity.Binding reagents may be selected from among any molecules known in theart to be capable of, or putatively capable of, specifically binding ananalyte of interest. The analyte of interest may be selected from amongthose described in Section 5.10 infra, “ECL Assays That May BeConducted.” Thus, the binding reagents include but are not limited toreceptors, ligands for receptors, antibodies or binding portions thereof(e.g., Fab, (Fab)′₂), proteins or fragments thereof, nucleic acids,oligonucleotides, glycoproteins, polysaccharides, antigens, epitopes,cells and cellular components, subcellular particles, carbohydratemoieties, enzymes, enzyme substrates, lectins, protein A, protein G,organic compounds, organometallic compounds, viruses, prions, viroids,lipids, fatty acids, lipopolysaccharides, peptides, cellularmetabolites, hormones, pharmacological agents, tranquilizers,barbiturates, alkaloids, steroids, vitamins, amino acids, sugars,nonbiological polymers, biotin, avidin, streptavidin, organic linkingcompounds such as polymer resins, lipoproteins, cytokines, lymphokines,hormones, synthetic polymers, organic and inorganic molecules, etc.Nucleic acids and oligonucleotides can refer to DNA, RNA and/oroligonucleotide analogues including but not limited to: oligonucleotidescontaining modified bases or modified sugars, oligonucleotidescontaining backbone chemistries other than phosphodiester linkages (see,for example, Nielsen, P. E. (1995) Annu Rev. Biophys. Biomcl. Street. 24167-183), and/or oligonucleotides, that have been synthesized ormodified to present chemical groups that can be used to form attachmentsto (covalent or non-covalent) to other molecules (where we define anucleic acid or oligo(nucleotide) as containing two or more nucleic acidbases and/or derivatives of nucleic acid bases).

The PMAMS of the invention may have a plurality of discrete bindingdomains that comprises at least one binding domain that contains bindingreagents that are identical to each other and that differ in specificityfrom the binding reagents contained within other binding domains, toprovide for binding of different analytes of interest by differentbinding domains. By way of example, such a PMAMS comprises a bindingdomain containing antibody to thyroid stimulating hormone (TSH), abinding domain containing an oligonucleotide that hybridizes tohepatitis C virus (HCV), a binding domain containing an oligonucleotidethat hybridizes to HIV, a binding domain containing an antibody to anHIV protein or glycoprotein, a binding domain that contains antibody toprostate specific antigen (PSA), and a binding domain that containsantibody to hepatitis B virus (HBV), or any subset of the foregoing.

A PMAMS may have a plurality of binding domains that comprises at leastone binding domain that contains within it binding reagents that differin binding specificity, so that a single binding domain can bindmultiple analytes of interest. By way of example, such a PMAMS comprisesa binding domain that contains both antibody to a T cell antigenreceptor and antibody to a T cell surface antigen such as CD4.

A PMAMS may have a plurality of binding domains that comprises (i) atleast one binding domain that contains binding reagents that areidentical to each other and that differ in specificity from at least oneof the binding reagents contained within the other binding domains; and(ii) at least one binding domain that contains within it bindingreagents that differ in binding specificities. By way of example, aPMAMS is made that has (a) at least one binding domain that containsbinding reagents of a single identity, e.g., antibody to a T cellantigen receptor, e.g., a, β T cell antigen receptor or γ, δ T cellantigen receptor), thus allowing this at least one binding domain tobind all cells expressing this T cell antigen receptor; and (b) at leastone binding domain that contains two different binding reagents, e.g.,antibody to T cell antigen receptor and antibody to CD4, thus allowingthis at least one binding domain to bind CD4⁺ T lymphocytes expressingthat T cell antigen receptor (i.e., a subpopulation of T lymphocytes).

In another embodiment, at least one binding domain contains bindingreagents which are different molecules but which have the same bindingspecificities (e.g., binding reagents such as epidermal growth factorand antibody to the epidermal growth factor receptor).

A plurality of binding reagents can be chosen so that even though thebinding reagents are different and have different binding specificities,they recognize the same analyte (in an alternative embodiment, differentanalytes are recognized). For example, where the analyte is an analytethat has numerous binding moieties (e.g., a cell, which has differentcell surface antigens), different binding reagents that bind todifferent binding moieties will recognize the same analyte. As anotherexample, antibodies to different cell surface antigens on a single cellwill recognize the same cell. As yet another example, antibodies todifferent epitopes of a single antigen can be used as binding reagentsto recognize the antigen.

A plurality of binding reagents can be chosen so that a plurality ofbinding domains may be formed where such binding domains recognize thesame analyte but with different affinities. The use of such a PMAMsallows for the detection of an analyte over a greater range ofconcentrations (e.g., a high affinity binding domain may be saturatedwith analyte under conditions that do not saturate a lower affinitybinding domain).

In still a further embodiment, only binding reagent(s) that specificallybind a single analyte of interest are present in one or more bindingdomains. Alternatively, binding reagents that specifically bind morethan one analyte of interest are present in one or more binding domains(e.g., a cross-reactive antibody). In a particular design, bindingreagents can be used that bind a class of analytes, e.g., with similarcharacteristics.

Binding domains may also be incorporated into a PMAMS that containbinding reagents that are specific for a desired standard analyte andthat are utilized as an internal standard (e.g., a binding domain whichcan be contacted with a sample containing a defined quantity of ananalyte to which the binding reagents bind). Multiple binding domainscontaining-binding reagents specific for the same analyte(s) can also beincorporated into a PMAMS so as to allow statistical averaging ofanalytical results. The binding reagents may not only be specific forthe same analyte, but may be identical, thus recognizing the samebinding moiety on the analyte. Thus, a plurality of binding domains(e.g., within a range of 2 to 10⁸) can be prepared that specificallybind to the same binding moiety, so that the ECL readings can bestatistically averaged to control for variation and improve accuracy.The plurality of binding domains on a PMAMS may be specific for acontrol analyte or an analyte of interest, or both, on a single support.

As another example, one or more discrete binding domains may be preparedwith a known initial concentration number of ECL labels. The built-inECL layer serves as a control to monitor, e.g., label degradation andtemperature effects.

A binding reagent may be used that is an enzyme specific for a substrate(said substrate being the analyte of interest), in which a product ofthe enzymatic reaction upon the substrate is a reporter agent (an agentthat is detectable), e.g., a product that triggers an ECL reaction, afluorescent molecule, a substance that changes color upon contact withappropriate enzyme (e.g., a chromogenic substrate for horseradishperoxidase), etc. In an example of such an embodiment, the enzyme usedas a binding reagent is glucose dehydrogenase (GDH), which can be usedto detect or measure glucose in a sample. An ECL label is situatedwithin or near to the binding domain containing the GDH. NADH isproduced by the action of the enzyme upon glucose, NADH being capable ofreacting with the ECL labels to promote ECL (Martin et al., 1993, Anal.Chim. Acta 281:475).

Binding domains containing binding reagents which increase backgroundbinding (i.e., that bind to a binding moiety present on the analyte ofinterest as well as on other analytes in the sample) can be used toincrease signal to noise ratios during the detection or measurement ofelectrochemiluminescence. By way of example, where the analyte ofinterest is a specific cellular subpopulation (e.g., CD4⁺ cells) and thesample is a fluid sample (e.g., blood) that contains cells from apatient, antibody to sialic acid can be used as a binding reagent toincrease background binding to virtually all cells in the sample (sincesialic acid is a component of virtually all cell surface glycoproteins),and an antibody to a cell surface antigen specific to the cellularsubpopulation (e.g., antibody to CD4) can then be used as a bindingreagent (in the same or different binding domain as that containing theantibody to sialic acid).

5.3. Voltage Waveform

The voltage waveform (change in electrical potential/time) impressedupon the electrodes and counter-electrodes of ECL cells must besufficient to trigger an ECL reaction. This-voltage waveform usually isin the form of a uniform voltage sweep starting at a first voltage,moving steadily to a second voltage, moving back through the firstvoltage to a third voltage and then back again to the first voltage. Forexample, the waveform may start at a first voltage in a range from −0.5through 0.5 volts, up to a second voltage in a range from 1 through 2.5volts and moving back through the first voltage to a third voltageranging from 0.0 to −1 volts. As another example, in simpler waves, thevoltage can be modified from 0.0 to +3.5 to 0.0. The voltage waveformsmay incorporate linear ramps, step functions, and/or other functions.The voltage waveforms may incorporate periods of time when the voltageremains fixed at one potential. The applied potential may be controlledrelative to one or more reference electrodes, or, no referenceelectrodes may be used. Additionally, negative potential may be used.Thus, the voltages used to induce ECL emissions from the cassette of thepresent invention will be readily selected for optimal ECL signalintensity and specificity for the ECL label and assay medium.

In some applications, the voltage is preferably varied as the lightemitted from the binding domain is measured. This is particularlyimportant to determine the threshold value of the electrical fieldnecessary to cause the binding domain to emit light. In this case, theelectrical potential applied at the binding domain starts at a valuebelieved to be below the threshold required to emit light, and a firstmeasurement is made of the light emitted. If no light is measured, orthe light is below a predetermined threshold, the electrical potentialapplied across the electrode pair is increased under computer control,such as by a computer controlled voltage source and another lightmeasurement is made. This process can be repeated until thepredetermined appropriate amount of light is received. In this way, thevoltage applied may be used as the assay signal.

The voltage waveform may contain an AC component. The use of such awaveform allows for better signal to noise in the detection of the ECLsignal, e.g., by filtering out signals that differ in frequency from thevoltage input).

The ordinary artisan who is familiar with the voltage and currentsettings as disclosed, for example, by U.S. Pat. Nos. 5,324,457 and5,068,088 will readily be able to select the optimum operating voltagesand voltage sweep for triggering ECL emission.

The potential required for generating ECL may be generated byillumination of the working electrode surface if the working electrodeis a semiconductor or contains another moiety that generates electricalcurrent in response to light.

5.4. Addressable Electrodes and Methods for Using the Same

Numerous methods may be used for addressing the plurality ofelectrode/counterelectrode pairs. Several illustrative such techniquesare illustrated in FIGS. 14-18. Shown in those figures by way of exampleare four electrode/counterelectrode pairs 101, 102, 103, 104 and awaveform generator which typically is a digital computer and whichpreferably is the same computer used for processing the ECL detected bythe detection means.

In FIG. 14, each electrode/counterelectrode pair 101-104 is individuallyaddressed by a pair of lines connected to the waveform generator. By wayof example, lines 105, 106 access electrode/counterelectrode pair 101.An appropriate waveform may be applied by the waveform generator at anygiven time to any one or more of the pairs of lines connected to thevarious electrode/counterelectrode pairs.

To reduce the number of connections required to address the electrodepairs, alternatives to the direct connection scheme of FIG. 14 areprovided. For example, a row-and-column accessing scheme is illustratedin FIG. 15 for electrically energizing some or all of the electrodes. Inthis scheme, one of the electrodes 201, 202 in each column of theplurality of electrode/counterelectrode pairs is connected to a commonelectrical conductor 205 on support 200, and each of thecounterelectrodes in each row of the plurality ofelectrode/counterelectrode pairs is connected to conductor 207, 208 onthe support 200. Conductors 205, 206 connect to connections C1, C2,respectively, at the edge of support 200 and conductors 207, 208 connectto connections R1, R2, respectively. Each of these connections is thenconnected by a separate line to the waveform generator. As a result, inthe configuration of FIG. 15, the number of required connections andsignal lines from the waveform generator has been reduced from 8 to 4.

To enable rapid and sequential energizing of each electrode pair, acomputer controlled switching device is beneficial. The configuration ofFIG. 16 shows a plurality of electrodes connected to a first multiplexer310. A plurality of counterelectrodes are connected to a secondmultiplexer 320. The first multiplexer is also connected to a first poleof a voltage source 330 that typically supplies the time varyingelectrical potential described infra. The second multiplexer is alsoconnected to a second pole of the voltage source. Using addressing linesA0-A3 electrically connected to each of the multiplexers and connectedto latch 340, a computer processor 350 can direct the multiplexers toselectively connect any or all of the first electrodes to the first poleof the voltage source, and any or all of the second electrodes to thesecond pole of the voltage source.

As shown in FIG. 17, a plurality of voltage sources are connectedthrough separate sets of multiplexers to each of the electrodes. If afirst electrical potential or range of electrical potentials is requiredat a particular electrode pair, the multiplexers 410, 420 associatedwith the voltage source 430 providing that potential are addressed bythe computer processor 350, typically through a latch 340, therebyconnecting that particular voltage source to the electrode pair inquestion. If a different electrical potential or range of electricalpotentials is required for another electrode pair, the multiplexers 440,450 associated with that different voltage source 460 are addressed bythe computer processor, thereby connecting that voltage source throughthe associated multiplexers 440, 450 to the electrode pair.

If the electrode array in this embodiment has at least a portion of theelectrode pairs independently driveable, as shown in FIG. 14, or 15, forexample, one electrode pair can be driven by one voltage source whileanother electrode pair is simultaneously driven with another voltagesource. Alternatively, the two voltage sources of FIG. 17 can bereplaced with a single voltage source connected to both sets ofmultiplexers in parallel, allowing two electrode pairs to be driven fromthe same voltage source.

Instead of a duplicate set of multiplexers for each voltage source asshown in FIG. 17, a plurality of voltage sources 520, 530 can beprovided as shown in FIG. 18. These voltage sources can be connectedthrough a computer controlled electrical switch 510 or switches to asingle set of multiplexers 310, 320. As shown in FIG. 18, the computerwould direct switch 510 to connect a particular voltage source to themultiplexers, and would also direct the multiplexers (by signallingtheir address lines A0-A3) to connect the selected voltage source to theparticular electrode pair desired.

Alternatively, the electrical potential applied to each of the electrodepairs in any embodiment can be varied. This is of particular benefitwhen a cassette having a plurality of different binding domains is used.Such a cassette may require a different range of applied electricalpotential at different binding domains. Several different embodimentscapable of varying the electrical potential applied to each electrodeare contemplated.

Advantageously, a computer controlled voltage source may be used. Acomputer controlled voltage source is one that can be addressed by acomputer to select a particular electrical potential to be supplied.Alternatively it can be programmed to sequentially apply a particularrange of electrical potentials over a predetermined time. In such asystem, address lines electrically connected to the computer and thevoltage source would allow the computer to program the voltage source toproduce the particular electrical potential to be applied to theelectrode pair to be energized.

Additional methods for addressing the plurality of electrode pairs mayalso be used. For example, a plurality of reference electrodes may beplaced in proximity to each of the plurality of electrode andcounterelectrode pairs in order to sense the voltage applied thereto. Inthis way, additional control of the voltage waveform may be maintained.

FIG. 36 shows another embodiment of the invention; arrays of electrodes(3600, 3601) are supported on each of two surfaces (3602, 3603)separated by a pattern of gaps in an insulator 3604 (for example aplastic sheet with punched holes 3605. Each electrode may pass over aplurality of gaps. If a potential is applied between one electrode oneach surface, current can only pass through a gap contacting bothelectrodes, thus limiting the location of any electrochemistry or ECLwhich may occur. In the preferred embodiment shown in the figure, theelectrodes (3600, 3601) are arrays of lines on a support. The two setsof electrodes on the two surfaces are oriented perpendicular to eachother. Gaps in the insulating sheet are located only at the intersectionof the electrodes from each surface.

This embodiment has the advantage over individually addressed electrodepairs that less electrical leads are required.

In an alternate embodiment, the insulator 3604 is omitted and thesurfaces are placed in close proximity so that only a narrow gap existsbetween the two surfaces. In this embodiment, a potential appliedbetween are electrode on each surface will preferentially cause currentto pass at the intersection of the electrode (i.e., where the distancebetween the electrodes is minimal) thus limiting the location of anyelectrochemistry or ECL which may occur.

5.5. Light Detection

The light generated by the triggered ECL emission is detected by anappropriate light detector or detectors positioned adjacent to theapparatus of the invention. The light detector may be, for example,film, a photomultiplier tube, photodiode, avalanche photo diode, chargecoupled device (“CCD”) or other light detector or camera. The lightdetector may be a single detector to detect sequential emissions or maybe plural to detect and spatially resolve simultaneous emissions atsingle or multiple wavelengths of emitted light. The light emitted anddetected may be visible light or may be emitted as non-visible radiationsuch as infrared or ultraviolet radiation. The detector or detectors maybe stationary or movable. The emitted light or other radiation may beconducted to the detector or detectors by means of lenses, mirrors andfiberoptic light guides or light conduits (single, multiple, fixed, ormoveable) positioned on or adjacent to the binding surface of thecassette or the detector may receive the light directly. In addition,the supports, PMAMS and electrode surfaces themselves can be utilized toguide or allow transmission of light.

The PMAMS may be formed on the surface of an array of light detectors sothat each detector only receives light from one binding domain. Thearray of light detectors may be a CCD chip, and the binding domains maybe attached (using standard coupling chemistries) to the surface of thesemiconductor device.

Drops deposited on the binding domains, or on a near, second surface,can be used as microlenses to direct or control emitted light.Alternatively, a light detector can be oriented directly in front of thecassette; and various light focusing devices, such as parabolicreflectors or lenses may be employed instead of a light conduit todirect light from any of a plurality of binding domains to the detector.The light emitted from at least two discrete binding domains may bemeasured simultaneously or sequentially.

Error due to thermal drift, aging of the apparatus, or the electricalnoise inherent in light detectors may be controlled by a “chopper” meansbetween the light measuring device and the binding domain beingmeasured. The chopper can be any one of the common mechanical chopperswell known to those of ordinary skill in the art, such as a spinningdisk with slots or cutouts that allow light to pass. Alternatively, thelight can be chopped by an LCD shutter, an array of LCD shutters, asolid state light valve or valves or the like. Alternatively, a planararray of LCD shutters or solid-state light valves such as those known inthe art of optical computing may be used. These devices are preferablylocated between the plane of the cassette, and the light conduit (orconduits) or light focusing devices that direct light from the bindingdomains to the light detector. In an embodiment, a shutter is locatedabove each of the binding domains. When using an LCD shutter or lightvalve, the shutters may be modulated at different frequencies tosimultaneously provide different chopping rates for different lightemitting binding domains. Using this technique, a plurality of differentlight signals may be superimposed and simultaneously measured by asingle detector. An electronic band pass filter electrically connectedto the light detector may then be used to separate the electrical singlesignal into several electrical components, each corresponding to one ofthe plurality of individual light components. By chopping the light, asabove, or using other mechanism well known to the art, an AC lightwaveform is created that allows the DC noise component to beelectronically filtered out.

Also, the ECL signal may be calibrated by comparison to resultspreviously determined with standard reagents to correct for signalmodulation due to reagent depletion.

5.6. Analysis of ECL signals

Signals arising from a given binding domain can have a range of values,and these values correlate with quantitative measurement to provide an‘analog’ signal. In another technique a ‘digital’ signal is obtainedfrom each domain to indicate that an analyte is either present or notpresent.

Statistical analysis is used for both techniques, and is particularlyuseful for translating a plurality of digital signals so as to provide aquantitative result. Some analytes, however, require a digitalpresent/not present signal indicative of a threshold concentration.‘Analog’ and/or ‘digital’ formats may be utilized separately or incombination. Other statistical methods can be utilized with PMAMS. Forinstance it is possible to create concentration gradients of PMAMS on asurface (Chaudhury et al., 1992, Science 256:1539-1541). This techniqueis used to determine concentrations through statistical analysis ofbinding over the concentration gradient. Multiple linear arrays of PMAMSwith concentration gradients may be produced with a multiplicity ofdifferent specific binding reagents. The concentration gradients mayconsist of discrete binding domains presenting different concentrationsof the binding reagents.

The presence of control assay systems on the binding surface of thecassette is also important to assure the uniformity of each analysis tocontrol for signal variation (e.g., variations due to degradations,fluctuations, aging of the cassettes and other components, thermalshifts, noise in electronic circuitry and noise in the photodetectiondevice, etc.). For example, multiple redundant binding domains(containing identical binding reagents or different binding reagentsthat are specific for the same analyte) for the same analyte may beutilized. In another example, analytes of known concentration areutilized or control domains of a PMAMS are covalently linked to a knownquantity of an ECL label or a known quantity of ECL label in solution isused.

The assays conducted according to the invention will rapidly andefficiently collect large amounts of data that can be stored, e.g., inthe form of a database consisting of a collection of clinical orresearch information. The data collected may also be used for rapidforensic or personal identification. For example, the use of a pluralityof nucleic acid probes when exposed to a human DNA sample can be usedfor a signature DNA fingerprint that can readily be used to identifyclinical or research samples.

5.7. Preparation of Electrodes for Multi Arrays

The electrodes may be broadly from 0.001 to 10 mm in width or diameter.In a preferred range the electrodes are from 0.01 to 1 mm in dimension(width or diameter or widest dimension depending upon the geometry ofthe electrodes).

Preferably, the electrodes are fabricated from suitable conductivematerials, such as transparent metal films or semiconductors (e.g., goldor indium-tin oxide, respectively), as is well known to the art, forexample, for the fabrication of liquid crystal displays and the like. Inthe assembled form of the cassette, sufficient space remains between thefirst and second supports to contain an analytic sample as, for example,a thin film or a wetted surface.

The electrodes may be fabricated from materials that contain carbon,e.g. particulate carbon, carbon black, carbon felts, glassy carbon,carbon fibers, carbon fibrils and/or aggregates of the above.

One or more individual fibrils and/or one or more aggregates of fibrilsmay be processed to form a larger aggregate (U.S. Pat. No. 5,124,075).In one embodiment, this larger aggregate is a mat or mesh. Hereinafter,the term “fibril mat” will be used to describe a mat or mesh of fibrilswhich the fibrils may be entangled interwoven. Fibril mats can have asurface area between 50 and 400 M²/gram.

By way of example, a fibril mat may be used as a working electrode, acounter electrode or a reference electrode in analytical and/orpreparative electrochemistry. In one example, the fibril mat is used asan electrode for electrochemiluminescence (ECL).

The binding domains of the PMAMS may be supported by an electrode, e.g.a fibril mat or an electrode formed from carbon black. The PMAMS of theinvention has a plurality of discrete binding domains, of which two ormore may be identical to each other or may differ. The fibril matsupports one or more binding domains.

One or more microfluidic guides may be used to prepare a plurality ofbinding domains on a fibril mat. Different or identical binding reagentsmay be present in a plurality of microfluidic guides and/or multipledistinct binding agents may be present in a microfluidic guide.

In FIGS. 22A and 22B a plurality of microfluidic guides 2201, preferablyin an array, are used to deliver, preferably concurrently, onto regionsof the fibril mat 2200, drops containing the desired binding reagents,to form discrete binding domains 2202. The binding reagents form a bondwith moieties present on the fibril mat. The binding reagents may adsorbnon-specifically to the mat or dry on the surface.

The desired binding reagents are delivered to the fibril mat whilesuction filtration is applied to the mat. In this instance, the suctionfiltration draws none, some or all of the binding reagents into orthrough the mat, and in doing so, reduces the amount of lateralspreading of the binding reagents on the surface of the mat during thepatterning process.

Fibril mats are prepared by compressing suspensions of carbon fibrilsonto a substrate through which the liquid of the suspension may pass(e.g., a filter). Examples of filters that may be used to form fibrilmats include filter paper, filters formed from polymeric (e.g., nylon)membranes, metal micromeshes, ceramic filters, glass filters,elastomeric filters, fiberglass filters and/or a combination of two ormore of such filter materials. One of skill in the art of filtrationwould recognize that these materials are merely examples of the manypossible materials suitable for filtration of suspensions of solids.

FIG. 23A and 23B illustrates an embodiment, in which fibril mats may befabricated by suction filtration. A dispersion and/or suspension ofcarbon fibrils 2301 is filtered using a filter 2300 equipped optionallywith a filter membrane 2303 and/or a filter support 2302. The suspensionis filtered using suction applied by a vacuum source 2305 to the filterby, for example, a filter flask 2306. A fibril mat 2304 collects oneither or both the filter membrane 2303 and/or the filter support 2302.The fibril mat 2304, with or without the filter membrane 2303 may beremoved from the filter.

In another embodiment, suspensions of fibrils are forced through afilter by use of pressure. In one example, pressure is exerted on aconfined suspension of fibrils by compressing a confined layer of airand/or liquid above the suspension with a piston. In a specific example,the suspension of fibrils is confined in a syringe, the piston is asyringe plunger, and the filter is a disposable syringe filter (manysuch filters are well known to one of skill in the art).

Suspensions of fibrils are forced through a filter by capillary actionor filtered by wicking of the suspension into or through a filter.

In another embodiment, individual fibrils or aggregates of fibrils arecrosslinked covalently into mats. Fibrils derivatized withphotosensitive moieties that polymerize when exposed to light areirradiated with light.

In another embodiment, individual fibrils or aggregate of fibrils aresprayed onto a substrate. It is possible to use electrospray.

The filter may be used to trap the fibrils in its pores and so form amat in which the filter acts as a support. In FIG. 24, a fibril mat 2400may be prepared by passing a slurry of fibrils 2401, delivered by asource 2402, between two large rollers 2403. In this process, which maybe analogous to processes found in the fabrication of paper or polymersheets, the rollers force the liquid from the suspension and a large,continuous mat of fibrils is produced from which smaller mats may becut.

Fibril mats may be freestanding (e.g., unsupported) or supported.

The rate of filtration can be varied to achieve desired properties inthe mat. For example, properties that may be varied include uniformityor non-uniformity of structure, the extent of entanglement of thefibrils or aggregates of fibrils, the thickness, the porosity of themat, and/or combinations thereof.

Suspensions of carbon fibrils are confined and the liquid in which thefibrils are suspended is removed. In one example, the liquid in whichthe fibrils are suspended is allowed to evaporate. In another example,the liquid is removed by heating. In yet another example, the suspensionis subjected to centrifugation, and the resulting liquid (e.g., thesupernatant) is removed. In another example, the liquid is removed byevacuation.

The suspension may be placed on one or more of the filters describedsupra, and the suspension dried by evaporation. The suspension may bedried by heating or baking in an oven or the liquid may be removed byfreezing and extracting the liquid. In yet another example, the liquidis removed by evacuation with a pump. Many other methods which are wellknown to one skilled in the art are available for removing liquids froma suspension.

Suspensions of fibrils suitable for forming fibril mats by filtrationmay be formed by dispersing one or more carbon fibrils in an appropriateliquid, quasi-solid or gel. Examples of appropriate liquids include butare not limited to water, ethanol, methanol, hexane, methylene chloride,buffered solutions, surfactants, organic solvents, solutions ofcontaining biological media (e.g., as proteins, antibodies or fragmentsthereof, cells, subcellular particles, viruses, nucleic acids, antigens,lipoproteins, liposaccharides, lipids, glycoproteins, carbohydrates,peptides, hormones or pharmacological agents, solutions of smallmolecules, polymer precursors, solutions of acids or bases, oils and/orcombinations thereof).

A suspension of fibrils may be prepared by dispersing carbon fibrils inan aqueous solution by means of sonication. In another embodiment,surfactant and or detergent may be added.

The fibril mat may have broadly a thickness between 0.01 μm and 10,000μm.

In preferred embodiments, the fibril mat has a thickness between 1 μmand 100 μm. In particularly preferred embodiments, fibril mats rangefrom 10 mm to 200 mm in width or diameter.

The fibril mat may be washed repeatedly and refiltered to removeresidual materials remaining from the suspension.

Fibril mats prepared by filtration or evaporation are heated (e.g., inan oven) to remove residual liquid from the suspension not removed byfiltration.

Successive filtration steps may be used to form mats of fibrils composedof one or more distinct layers that are either in contact with or inclose proximity to one or more other layers. Layers may be distinguishedby several properties, including but not limited to differences in theporosity, the density, the thickness, the distribution of sizes ofindividual fibrils and/or microscopic aggregates of fibrils, the type,number and/or size of fibril aggregates, the chemical derivatization ofthe fibrils (vide infra), and/or the presence of other matter attachedto the fibrils.

FIG. 25, a multi-layered fibril mat 2500 is prepared by successivefiltration steps. A 0.5 μm to 100 μm thick layer 2501 of plain fibrilsforms the first layer; a 0.5 to 10 μm thick layer of fibrils 2502 thatincorporate moieties such as poly-(ethyleneglycols) that resistadsorption of proteins and other molecules forms the second layer; a 0.5to 5 μm thick layer 2503 that incorporates one or more binding domains(vide supra) forms the third layer. The binding domains contain one ormore antibodies 2504, which may bind an analyte 2505. Thisantibody/analyte complex may bind a labeled antibody 2506. The label maybe an ECL label. In other embodiments, the label may be one or more of aplurality of labels described elsewhere in this application. Such amultilayer mat may be freestanding or supported on one of a plurality ofpossible supports described above.

Multilayer mats may be formed in which there are combinations of layers,in which some or all of the layers may be different.

The filter used to form the fibril mat, the fibrils, and/or the fibrilmat may be coated. In particular embodiments, the coatings are metallic.These coatings may be patterned such that certain portions are coated,and other portions are not. In one example, the coating is applied byelectrodeposition. In another example, the coating is applied byelectroless deposition. Other methods of metal deposition, e.g. thermalor electron bean depositions or sputtering, can also be used.

The filter is coated with a metal, and the fibril is derivatized with achemical functional group that forms a bond with said metal. The filteris a metal screen or metal sheet.

The fibril mat may be flat or deformed, regular or irregular, round,oval, rectangular, or one of many shapes, rigid or flexible,transparent, translucent, partially or fully opaque and may havecomposite properties or regions of different individual or compositeproperties.

The mat may be a disk or a piece taken from a sheet.

A plurality of fibril mats may be fabricated, preferably concurrently,and preferably in an array. In-one example, an array of microfluidicguides forms a plurality of fibril mats on a support as described above.In another example, an array of filters, or a patterned filter (e.g.,with regions of different porosity) is used to prepare an array offibril mats.

A mask with an array of holes (e.g., a screen) is used to cover certainportions of a filter or support, and a plurality of discrete fibril matsare made concurrently by either filtration and/or evaporation.

Fibril mats may have a density from 0.1 to 3.0 grams/cm². The mat mayhave variable density. For example, mechanical force or pressure may beapplied to the mat at different times to increase or decrease thedensity.

Fibril mats may have pores. These pores may extend partially and/orfully through the mat or may be part of a network or pores. These poresmay have dimensions ranging broadly from 50 Å to 1000 μm. In a preferredembodiment, the fibril mat has pores with dimensions ranging from 200 Åto 500 Å. The porosity of the mat may depend on the density of the mat,among other factors.

The porosity of the mat may be constant throughout the mat or mayincrease or decrease as a function of the position in the mat. Thefibril mat may have a wide variety of pores of different sizedistributed in a disorganized and/or random manner.

The fibril mat may contain distinct regions of different porosity. Forexample, the fibril mat may have one or more layers, each having adifferent porosity. The fibril mats may have columns of differentporosity that run through the mat.

The porosity of the mat may be varied by including different amounts ofaggregates of carbon fibrils, where aggregates have different size,shape, composition, or combinations. In a particular example, a mat canbe prepared from individual fibrils, CC fibrils (described supra) and BNfibrils (described supra), or different combinations. For example, thefibril mat may have some pores that are large enough to pass objects aslarge as biological cells, some pores that can pass biological media aslarge as proteins or antibodies, some pores that can pass only small(<1000 molecular weight) organic molecules, and/or combinations thereof.

The porosity of the mat may be such that one or more molecules, liquids,solids, emulsions, suspensions, gases, gels and/or dispersions candiffuse into, within and/or through the mat. The porosity of the fibrilmat is such that biological media can diffuse (actively or passively) orbe forced by some means into, within and/or through the mat. Examples ofbiological media include but are not limited to whole blood,fractionated blood, plasma, serum, urine, solutions of proteins,antibodies or fragments thereof, cells, subcellular particles, viruses,nucleic acids, antigens, lipoproteins, liposaccharides, lipids,glycoproteins, carbohydrates, peptides, hormones or pharmacologicalagents. The fibril mat may have one or more layers of different porositysuch that material may pass through one or more layers, but not throughother layers.

The fibril mat is supported by or on another material. By way ofexample, the supporting material may be a metal, plastic, polymer,elastomer, gel, paper, ceramic, glass, liquid, wax, oil, paraffin,organic solid, carbon or a mixture of two or more of each. The materialmay be solid or liquid. If it is solid, it may contain one or aplurality of holes or pores. In specific examples, the support may be ametal mesh, a nylon filter membrane or a filter paper. The support maybe a conductor, a semiconductor and/or an insulator. The fibril mat mayincorporate another material, for example thin fibers, shards, or ballsof metal to increase the conductivity of the mat. In another example,the fibril-mat may incorporate-other carbon, glass and/or metal fibersof varying size, shape and density to create a different porosity thancan be achieved with fibrils alone. In another aspect, the mat mayincorporate magnetic beads (for example, DYNAL beads). In the latterexample, the beads may either serve to change a property of the mat, ormay themselves be used as supports to immobilize binding domains.

In an embodiment disclosed in U.S. Pat. Nos. 5,304,326 and 5,098,771,fibrils may be dispersed in another material. For example, fibrils maybe dispersed in oils, waxes, paraffin, plastics (e.g., ABS, polystyrene,polyethylene, acrylonitrile, etc.), ceramics, teflon, polymers,elastomers, gel, and/or combinations thereof. Dispersions of fibrils inother materials are conducting. Dispersions of fibrils in othermaterials may be molded, pressed, formed, cast, spun, weaved, and/orthrown so as to form objects of a desired shape and/or form.

Other carbon materials (e.g., particulate carbon, carbon fibers,graphitic carbon, buckminsterfullerenes, fullerenes, or combinationsthereof) may be dispersed in another material. They can be derivatizedwith chemical functional groups that can be used to attach othermaterials to them. Materials may be attached covalently to thesefunctional groups, or they may be adsorbed non-covalently.

Carbon fibrils may be prepared with chemical functional groups attachedcovalently to their surface. As described in International PublicationNo. WO 90/14221, these chemical functional groups include but are notlimited to COOH, OH, NH₂ N-hydroxy succinimide (NHS)-esters,poly-(ethylene glycols), thiols, alkyl ((CH₂)_(n)) groups, and/orcombinations thereof. These and other chemical functional groups can beused to attach other materials to the surface of fibrils.

Certain chemical functional groups (e.g., COOH, NH₂, SH, NHS-esters) maybe used to couple other small molecules to the fibrils. There are aplurality of possible combinations of such chemical functional groupsand small molecules.

In many embodiments, NHS-ester groups are used to attach other moleculesor materials bearing a nucleophilic chemical functional group (e.g., anamine). In a preferred embodiment, the nucleophilic chemical functionalgroup is present on and/or in a biomolecule, either naturally and/or bychemical derivatization. Examples of suitable biomolecules include butare not limited to amino acids, proteins and functional fragmentsthereof, antibodies, binding fragments of antibodies, enzymes, nucleicacids, and combinations thereof. This is one of many such possibletechniques and is generally applicable to the examples given here andmany other analogous materials and/or biomolecules. In a preferredembodiment, reagents that may be used for ECL may be attached to thefibril via NHS-ester groups.

An antibody that can be used in an ECL assay can be attached to one ormore fibrils or a fibril mat by covalent bonds (e.g., reaction with anNHS-ester), by reaction with an appropriate linker (vide supra), bynon-specific binding, and/or by a combination thereof. Nucleic acidsand/or cells can be attached to fibrils or fibril mats by covalent linksto NHS-esters attached to the fibrils.

It may be desirable to control the extent of non-specific binding ofmaterials to fibrils and/or fibril mats. Simply by way of non-limitingexamples, it may be desirable to reduce or prevent the non-specificadsorption of proteins, antibodies, fragments of antibodies, cells,subcellular particles, viruses, serum and/or one or more of itscomponents, ECL labels (e.g., Ru^(II)(bpy)₃ and Ru^(III)(bpy)₃derivatives), oxalates, trialkylamines, antigens, analytes, and/orcombinations thereof. In another example, it may be desirable to enhancethe binding of biomolecules.

One or more chemical moieties that reduce or prevent non-specificbinding may be present in, on, or in proximity to a carbon-containingelectrode (e.g. carbon black) or one or more fibrils, one or more fibrilaggregates, a dispersion of fibrils in another material and/or a fibrilmat. Such moieties, e.g., PEG moieties and/or charged residues (e.g.,phosphates, ammonium ions), may be attached to the electrode.

Materials used in the support, electrode and/or binding domain may betreated with surfactants to reduce non-specific binding. For example,fibrils or fibril mats may be treated with surfactants and/or detergentsthat are well known to one of ordinary skill in the art (for example,the Tween series, Triton, Span, Brij). The fibrils or fibril mats arewashed, soaked, incubated with, sonicated in, and/or a combinationthereof with solutions of surfactants and/or detergents. Solutions ofPEGs and/or molecules which behave in similar fashion to PEG (e.g.,oligo- or polysaccharides, other hydrophilic oligomers or polymers)(“Polyethylene glycol chemistry: Biotechnical and biomedicalapplications, Harris, J. M. Editor, 1992, Plenum Press) may be usedinstead of and/or in conjunction with surfactants and/or detergents.

Undesirable non-specific adsorption of certain entities such as thoselisted above may be blocked by competitive non-specific adsorption. Thiscompetitive binding species might be bovine serum albumin (BSA)immunoglobulin G (IgG).

Non-specific binding of the ECL-TAG may be reduced by chemicalmodification of the TAG. For example, the TAG may be modified so as toincrease its hydrophilicity (e.g. by adding hydrophilic, polar, hydrogenbonding, and/or charged functional groups to the bipyridyl ligands inRu(bpy₃)) and thus reduce non-specific binding of the TAG to othersurfaces.

It may be desirable to immobilize biomolecules or other media tocarbon-containing materials, e.g. carbon black, fibrils, a fibril mat,and/or carbon dispersed in another material. One may attach antibodies,fragments of antibodies, proteins, enzymes, enzyme substrates,inhibitors, cofactors, antigens, haptens, lipoproteins, liposaccharides,cells, sub-cellular components, cell receptors, viruses, nucleic acids,antigens, lipids, glycoproteins, carbohydrates, peptides, amino acids,hormones, protein-binding ligands, pharmacological agents, and/orcombinations thereof. It may also be desirable to attach non-biologicalentities such as, but not limited to polymers, elastomers, gels,coatings, ECL tags, redox active species (e.g., tripropylamine,oxalates), inorganic materials, chelating agents, linkers etc.

One or more or a plurality of species may become bound non-specifically(e.g., adsorb) to the surface of a material comprised of carbon.

Biological molecules or other media can be attached to fibrils or fibrilmats by non-specific adsorption. The extent of non-specific adsorptionfor any given fibril, fibril mat and/or biomolecule will be determinedby certain properties of each. Certain chemical functional groups orbiological moieties present on fibrils may reduce or enhancenon-specific binding. The presence of hydrophobic and/or hydrophilicpatches on the surface of a protein may enhance or reduce non-specificbinding of the protein to fibrils or fibril mats. Hydrophilic and/orhydrophobic patches are utilized to control non-specific binding incontrolled areas.

Carbon can be derivatized with alkyl (CH₂) chains and/or carboxylic acidgroups to enhance non-specific binding of biological molecules or mediaor other materials.

FIG. 26 illustrates the above embodiment schematically in the case of asingle fibril. A fibril 2600 is derivatized with alkyl chains 2601.Biomolecules 2602, 2603, and 2604 bind non-specifically to the alkylchains. Polymer/elastomer 2605 is also bound.

Underivatized fibrils, fibril aggregates and/or fibril mats are used forimmobilization of biomolecules, biological media, and other materials bynon-specific binding.

The ECL TAG may contains charged residues that could be used toselectively attract a TAG-labeled moiety to a support and/or electrode.For example, a derivatized ECL TAG which has a net negative charge mayhave a relatively low affinity for an electrode at more reducingpotentials and then have higher affinity for the electrode as theelectrode potential becomes more oxidizing. The affinity of the ECLlabel and/or binding reagents to the electrode may be made to modulate.This modulation may be used to improve the kinetics of binding orimprove the efficiency of a washing step.

In FIG. 28 molecules (both biological and non-biological) may beattached to fibrils by means of a covalent bond. Fibrils 2800 bearing anNHS-ester chemical functional groups may form covalent bonds 2801 tobiomolecules or biological media 2802,2803. These biological media mayuse an amino group to form a covalent bond by reaction with theNHS-ester group. Polymer 2808 is immobilized. One of ordinary skill inthe art would recognize the generality of NHS-ester groups as couplingagents for molecules and would be able to select both the appropriatebiomolecules and the appropriate reaction conditions to achieveimmobilization.

A pair of moieties and/or molecules “M1” and “S1”, of which one or moreis attached to a fibril, exhibit a mutual affinity or binding capacity.M1/S1 may be antibody/antigen, antibody/hapten, enzyme/substrate,enzyme/cofactor, enzyme/inhibitor, lectin/carbohydrate,receptor/hormone, receptor/effector, nucleic acid/nucleic acid,protein/nucleic acid, virus/ligand, cell/cellular receptor, etc. Manycombinations of “binding pairs” M1/S1 and would be able to selectcombinations appropriate to achieve the desired binding. Either or bothM1 and S1 may be attached to one or more fibrils.

FIGS. 27 and 28 illustrate some of the many possible configurations thatare possible with this embodiment. In FIG. 27, a fibril 2700 derivatizedwith alkyl chains 2701 non-specifically binds a molecule 2702 that has amutual affinity or binding capacity for another molecule 2703. Molecule2703 is also attached to another molecule 2704. A blocking molecule 2705may be non-specifically adsorbed to the fibril. A blocking polymer 2706and/or a polymer 2707 which has a ligand (2708) that has an affinity fora molecule 2709 are non-specifically adsorbed.

In FIG. 28, a fibril 2800 is covalently linked via 2801 to biomolecules2802 and 2803, and a linker molecule 2804. The linker molecule 2804 hasa mutual affinity or binding capacity for another biomolecule 2805.Biomolecule 2803 has a mutual affinity or binding capacity for anotherlinker molecule 2806, which is covalently linked to 2807. Polymer 2808with a ligand 2812 that is specific for a binding partner 2809 iscovalently linked to a fibril. Blocking molecules (e.g. BSA) 2811 andblocking polymers 2810 are covalently attached.

A fibril may be derivatized with biotin and/or a biotinylated linker andavidin and/or streptavidin may bind to this linker. Avidin and/orstreptavidin may be bound to the fibril, and a biotinylated antibodyand/or protein may bind. Avidin and/or streptavidin may be immobilizedon the fibrils by either non-specific binding, covalent bond, another orthe same coupling pair, or a combination thereof. The use of(strept)avidin and biotin as “binding pairs” is a widely applied methodof attaching biomolecules or biological media to other materials and iswell known to those skilled in the art (Spinke et al., 1993, Langmuir9:1821).

A binding pair may be a monoclonal antibody and an antigen that binds tothis antibody.

Multiple binding pairs (e.g., M1/S1/M2) may form. M1 is a monoclonalantibody, S1 is an antigen to M1, and M2 is an antibody that binds toS1. This complex may constitute an antibody/antigen/antibody “sandwich”complex (such antibodies may or may not be monoclonal). M2 may be anantibody tagged with an ECL-active tag (vide supra), a fluorescentlabel, a radioactive label, an enzymic tag, and/or combinations thereof.

M1 may be a moiety that can complex with a metal, metal ion, ororganometallic compound (a “chelating agent”) and S1 is a metal, metalion, or organometallic compound (a “chelates”) that forms a complex withM1, and M2 is a moiety on a biological molecule that binds to the M1/S1complex (Gershon and Khilko, 1995, Journal of Immunological Methods,7371).

The fabrication of metallic electrode patterns and conductive elementsto distribute electrical current to such electrodes on a surface iscarried out by methods well known to the art (see, e.g., Leventis etal., U.S. Pat. No. 5,189,549). The preparation of metal films ontransparent surfaces is used to produce liquid crystal displays and isreadily adapted to the preparation of electrodes according to theinvention. Haneko, 1987, Liquid Crystal TV Displays, Principles andApplications of Liquid Crystal Displays, KTK Scientific Publishers,Tokyo, D. Reidel Publishing. Transparent electrode surfaces may also beprepared, for example, according to the method of DiMilla et al., 1994,J. Am. Chem. Soc. 116(5):2225-2226. 0.5 nm of titanium and 5 nm of goldare deposited on transparent substrates (glass or plastic). A thin goldlayer as prepared by the method of DiMilla, supra may be used to preparea transparent electrical structure by the method of Kumar supra.Modifications of this procedure to increase the thickness of theconductor layers for improved current carrying capacity while preferablymaintaining transparency are desirable and readily apparent to theordinary artisan. Such techniques may be used to prepare electrodesurfaces that are aligned with or in proximity with discrete bindingdomains of a PMAMS.

In addition, the films and/or monolayers may be composed of moietieswhich facilitate the transfer of electrical potential from the electrodesurface to the ECL label, rather than using insulating moieties (e.g.,alkyl chains) as taught by Zhang and Bard. For example, pi orbitaloverlap in extensively conjugated systems can be used for electrontransfer. Such pi orbital electron transfer is provided by poly-pyroleor other conjugated rings or double bonded structures.

Oligonucleotides may be utilized to modulate electron transfer. Forexample, overlapping pi bonds in double stranded DNA may be utilized toincrease electron transfer rates. Oligonucleotides bound to an electrodesurface can be utilized as a binding agent in a binding domain. Uponbinding a complementary oligonucleotide sequence a double strand withorganized overlapping pi bonds is formed. In a particular embodiment, afirst or primary immobilized (e.g., covalently linked to a support)oligonucleotide is ECL labeled. In another embodiment a secondarycomplementary oligonucleotide or oligonucleotide of partiallycomplementary sequence to the primary oligonucleotide is ECL labeled. Atertiary oligonucleotide complementary to or partially complementary tothe secondary oligonucleotide is labeled (e.g., a sandwich assay).Branched oligonucleotide chains may also be utilized. A variety ofoligonucleotides and/or oligonucleotide mimics can be utilized (e.g.,oligonucleotides with modified bases and/or modified backbonescontaining for example nitrogen and/or sulfur). Differential studies maybe performed. Variable stability of pi overlap in oligonucleotidesand/or oligonucleotide complexes may be monitored through modulation ofelectron transfer. The signal (e.g., ECL light generated and/orimpedance measurements) generated from a pi bond stabilized ECL labeleddouble helical oligonucleotide pair may be correlated against the signaland/or expected signal from a more disordered single strandedoligonucleotide. The variation in ECL signal between a fullycomplementary ECL labeled double stranded oligonucleotide and apartially complementary ECL labeled double stranded oligonucleotide maybe correlated. Additionally, oligonucleotide complexes of multipleoligonucleotides may be utilized. For example, triple helices may beemployed.

Modulation of electron transfer rates may be measured using ECLdetection as well as electronic means. ECL labels may be covalentlylinked to oligonucleotide strands and/or non-covalently associated(e.g., intercalated). DNA may be coupled to the electrode without theuse of a linker (e.g., adsorption of 5′ thiolated DNA on gold) or with ashort (<10 atom) linker to ensure low resistance to electron transferfrom the DNA to the electrode. A linking chain may be used that canefficiently transport electrons from the electrode to the DNA strand(e.g., a polyacetylene chain).

A mixed monolayer and/or film may be used in which at least oneconstituent of the monolayer or film, as the case may be, facilitatesthe transfer of electrical potential. Alternatively, a molecule orparticle that facilitates the transfer of electrical potential isadsorbed to the monolayer or film. As examples of the foregoing, the piconjugated monolayers and/or conducting micro-particles which adsorb toand/or are approximate to the electrode surface, may be used. Patternedregular gaps are created in the monolayer and or film. By utilizingcontrolled patterns of gaps in an ordered substantially perpendicularSAM composed of long chain alkane thiols (i.e., insulating) to which ECLlabels have subsequently been attached, the effective potential imposedat the ECL labels can be controlled. For example, FIG. 11 shows acassette 1200 formed of a single support 1202 with a metallic layer1204, a SAM pattern 1206 and gaps 1208 between the SAM patterns.

ECL labeled proteins may be non-covalently linked to a monolayersurface. An ECL labeled protein may adsorb to the surface of a methylterminated alkane thiol derivatized gold surface. The gold surface mayact as the working electrode or the counter electrode. A plurality ofbinding domains may be incorporated on a single support as isillustrated in FIGS. 11-13. In preferred embodiments the binding domainscontain labelled and/or unlabelled proteins and/or nucleic acids and/orcells and/or chemical species.

Alternatively, the length of the components of the monolayer (e.g., thelength of the alkane chain in alkane thiol monolayers) may be varied tocontrol the effective potential at the exposed surface of the monolayer.

Broadly, alkane thiols may have carbon chains of length between 1 carbonand 100 carbons. In a preferred embodiment the carbon length of thealkane thiol contains between 2 and 24 carbons. The carbon chain lengthof the alkane thiol is between 2 and 18 carbons. The carbon chain lengthis between 7 and 11 carbons. Such alkane thiols may have a variety ofhead groups exposed to the assay media including methyl groups, hydroxygroups, amines, carboxylic acids, oligo (ethylene glycols), phosphategroups, phosphoryl groups, biotin, nitrilotriacetic acid, glutathione,epoxides, dinitrophenyl, and/or NHS esters. Other head groups includeligands commonly used for the purification and immobilization ofrecombinant fusion proteins (e.g., Sassenfeld, 1990, TIBTECH 8:88-93).The binding domains may be derivatized to varying degrees to achievevarying densities of binding reagents. For example, different densitiesof activatable chemistries may be used and/or derivatization may becarried out to varying extents. Mixed chemistries may be utilized tocreate desired binding densities. Mixed monolayers may be utilized tocontrol the density of activatable groups and/or binding reagents. Thedensity of binding groups is controlled to optimize the ECL signal tonoise ratio. The total number of binding sites within a bindingdomain(s) is controlled to optimize the intensity of the ECL lightsignal with respect to other ECL light signals from other bindingdomain(s) whether such ECL light signals are detected sequentially orsimultaneously and/or with respect to the light detection means.

The voltage waveform may be applied so as to activate ECL labelsassociated with a binding domain(s) within a PMAMS one or more times. Anelectronic potential sufficient to activate ECL light generation may beapplied multiple times to the same alkane thiol derivatized surface withbound ECL label to generate multiple ECL light signals. Electronicpotential is applied sufficiently to generate ECL reversibly. Potentialis applied so as to generate ECL quasi-reversibly. In a quasi-reversibleseries of voltage waveforms the binding domain within which ECL labelassociates (e.g., binds), may be chemically and/or physically altered.The voltage waveform series applied may yield irreversible ECL lightgeneration.

Further, an electric potential sufficient to release the components ofthe monolayer may be applied. It is desirable to release such monolayercomponents where the volume above the electrode surface is small (e.g.,another support or plate resting on the electrode surface). In this wayas the monolayer is disrupted, even some ECL labels that are notefficiently excited may be excited by the electrode surface to generatethe electrochemiluminescent signal and the ECL labels are restricted toa small volume restricting diffusion from the electrode. Variousmonolayer compositions may be utilized to control the degree ofmonolayer disruption for a given potential. Monolayers with componentswith strong inter-component affinity will be more resistive to monolayerdisruption. Longer alkane chain thiols resist disruption moreeffectively than short alkane chain thiols. By varying the chain lengththe desired stability may be achieved.

Modification of the binding domains within a PMAMS may be used tomodulate the ECL signal. A series of voltage waveforms is applied so asto generate a multiplicity of ECL signals. Said multiplicity of ECLsignals may be utilized to gain extra and/or better results. Statisticalanalysis of the rate of modulation of the ECL signal may be correlatedto the overall quality of one or more binding domains. Additionally,said multiplicity of ECL signals may be utilized to increase signal tonoise by, for example, filtering certain ECL signals of a series.Further, multiple electronic potential waveform pulses may be utilizedto reduce undesirable modulation of signal due to non-specific binding.Electronic potential may be applied to prevent non-specific binding ofcertain charged species. Additionally, electronic potential may beapplied so as to promote the localization near a binding domain(s) ofcertain analytes or chemical species of interest. The voltage waveformapplied supplies large over-potential (e.g., higher potential than isrequired to generate ECL). Over-potentials may be utilized to modulateECL signals in a voltage wave series or in a single voltage wave pulse.Additionally, over-potentials may be utilized to modulate the ECLreaction kinetics and/or modulate the binding potential chemicallyand/or physically. Further, one or more voltage waveforms and/or otherelectronic probes known to those skilled in the art may be utilized toassess and/or correlate and/or extrapolate information on the qualityand/or electronic properties of an electrode(s).

Preferably, the efficiency of the ECL reaction may be enhanced byextending the working electrode surface area by providing additionalconducting means in contact with the electrodes. Projections orextensions from the electrode (e.g., wires or whiskers) of conductingmaterials or conducting particles may be used to increase the electrodesurface area, such that the electrical field and more closely approachesthe ECL label. Alternatively, indentations or wells in the electrodestructures may serve the same purpose.

In particular, conductive particles may fill the gaps on the electrodesurface and/or cover the support or monolayer so that the electricalfield around the ECL label is increased in absolute magnitude, as shownby FIG. 12. These conductive particles extend the electrode surface areaand thereby increase the efficiency of the ECL reaction. FIG. 12 shows acassette 1300 having a support 1302 bearing a patterned SAM 1306 on ametallic layer 1304 and indicates conducting micro-particles filling inthe gaps (e.g., 1208 in FIG. 11) and extending above the metallicsurface between the SAM patterns. For magnetic conducting particles, amagnet or magnetic field may be used to draw the particles to thesurface. The conductive particles may also be used as described toextend the electrical potential between the electrode surfaces and thebinding domain of a PMAMS with two approximated supports. In FIG. 8, thecassette 900 consists of a first support 902 that has a multi array ofelectrodes, and a second support 904 that has a PMAMS. Conductingmicro-particles 906 are positioned between the opposing surfaces inorder to extend the electrical potential toward the ECL label on thebinding domains (not shown).

Alternatively, conductive polymers are grown from the exposed gaps onthe electrode surface to facilitate extending the electrical potentialaround the ECL label of the sample as shown by FIG. 13. FIG. 13 shows acassette 1400 having a support 1402 bearing a metallic layer 1404 on apatterned SAM surface 1406. Conductive polymers 1408 are grown over theSAM surface to extend the electrical field provided by a multi array ofelectrodes (not shown) to binding domains (not shown) on the SAMsurface. The conductive polymers may also be used as described to extendthe electrical potential between the electrode surfaces and the bindingdomains of a PMAMS of two approximated supports as illustrated by FIG.7. In FIG. 7, the cassette 800 consists of approximated supports 802 and804. Conductive polymers 806 are grown between the opposed surfaces soas to extend the electrical potential toward the ECL label on thebinding domain (not shown).

FIG. 9 illustrates a cassette 1000 formed with a first support 1002having a multielectrode array, a second support 1004 having a PMAMSbinding surface, wherein conductive projections (1006) (e.g., fine wireor other protrusions) of the working electrode extend the electricalfield around the ECL label in the PMAMS binding domains.

The electrode pairs may be created in a variety of configurations. Thesimplest configurations, depicted in the figures accompanying thisdisclosure, are made of metal and/or metal oxide films and/orsemiconductor films applied on a non-conducting planar surface. Theelectrodes of these electrode pairs preferably define between them aregion of relatively constant width thereby providing a relativelyconstant electrical field.

Other configurations of the electrodes are provided. Several of theseconfigurations are shown in plane views in FIGS. 19(a)-(e). FIG. 19(a)shows an inter-digitated comb-like electrode pair. In this structure,each electrode has a plurality of digits extending from the conductormaking a comb-like shape. The electrode and counterelectrode pair may bepositioned adjacent to a binding domain, or a binding domain may bepositioned between an electrode and counterelectrode. FIG. 19(b) shows apair of concentric electrodes, one circular and one semicircular. FIG.19(c) shows two semicircular electrodes with their straight edges facingeach other. FIG. 19(d) shows a pair of rectangular electrodes. FIG.19(e) shows a pair of interdigitated electrodes having complementaryopposing curved surfaces to form a sinuous gap therebetween.

The electrode/counterelectrode pairs may also be formed into specificshapes complementary to shapes on the PMAMS binding surface foralignment purposes. Exemplary shapes are shown in FIG. 6B. A support 712bearing electrode pairs 714-720 is shown. The electrode pairs may be,e.g., circular 714, interdigitated 716 triangular interdigitating 718 ormulti electrode interdigitating 720.

In the embodiments shown in FIGS. 14-19 discussed supra, the electrodepairs are located on a single support. Alternatively, the electrodepairs are located on first and second opposing supports as shown by FIG.2.

5.8. Cassettes

Cassettes contain one or more supports of the invention. Cassettes mayinclude a plurality of binding domains and one or more workingelectrodes.

FIG. 2 depicts a cassette where each of plural binding domains 30 onsupport 26 are adjacent to a different one of plural electrodes 32.Counterelectrodes 38 are formed on a second support 28. An ECL assay isconducted as previously described by placing a sample on binding domains30 and then moving together supports 26 and 28 so that counterelectrodes38 are each adjacent to each of binding domains 30 and an ECL reactionmay be triggered as described above by waveform generator means 39, viaa lead 34, and an ECL signal detected and recorded by light detectormeans 40, wire 41, and digital computer means 42.

FIG. 3 illustrates a cassette where each of plural binding domains 48has a different one of plural electrode/counterelectrode pairs 50adjacent thereto on support 44. Support 46 may optionally be placedadjacent to support 44 so that support 46 forms a sample containingmeans adjacent to plural binding domains 48 and plural electrodes 50.Thus, an ECL reaction may be triggered via electrical connection 52 bywaveform generator means 54, and an ECL signal detected by lightdetector means 56 and recorded and analyzed by digital computer means58.

A cassette is provided that contains one or more pairs of supports asshown in FIG. 21, each pair of supports being situated so that thesurface of a first support 1501 that contains binding domains faces thesurface containing binding domains on the second support 1502, in whicheach surface contains electrodes 1504 and binding domains 1506; suchthat each binding domain on the first support faces and is aligned withan electrode on the second support, and each binding domain on thesecond support faces and is aligned with an electrode on the firstsupport.

FIG. 4 illustrates a cassette wherein ECL electrodes are optional.Binding domains 64 on support 60 are contacted with a sample suspectedof containing an analyte. Regions 66 on support 62 contain reactionmedium for detecting or measuring an analyte of interest or for carryingout a desired reaction. Support 60 and Support 62 are brought togetherso that binding domains 64 and regions 66 are contacted and the presenceof an analyte or reaction product is determined by a reporter system,e.g. a calorimetric chemiluminescent or fluorescent signal that may bedetected by photodetector means 68 and recorded and analyzed by digitalcomputer means 70.

In a preferred embodiment, a cassette or apparatus of the inventioncomprises a means for sample delivery onto the plurality of discretebinding domains (see, e.g., element 1 on FIG. 1 of U.S. Pat. No.5,147,806; element 1 on FIG. 1 of U.S. Pat. No. 5,068,088; each of whichis incorporated by reference in its entirety). The means for sampledelivery can be stationary or movable and can be any known in the art,including but not limited to one or more inlets, holes, pores, channels,pipes, microfluidic guides (e.g., capillaries), tubes, spigots, etc.Fluids can be moved through the system by a variety of well knownmethods, for example: pumps, pipettes, syringes, gravity flow, capillaryaction, wicking, electrophoresis, pressure, vacuum, etc. The means forfluid movement may be located on the cassette or on a separate unit. Thesample can be placed on all of the binding domains together.Alternatively, a sample may be placed on particular binding domains by acapillary fluid transport means. Alternatively, samples may be placed onthe support by an automatic pipetter for delivery of fluid samplesdirectly to the PMAMS on a support, or into a reservoir in a cassette orcassette holder for later delivery directly to the binding surface.

Supports may be prepared from materials including but not limited to,glass, plastic, ceramic, polymeric materials, elastomeric materials,metals, carbon or carbon containing materials, alloys, composite foils,silicon and/or layered materials. Supports may have a wide variety ofstructural, chemical and/or optical properties. They may be rigid orflexible, flat or deformed, transparent, translucent, partially or fullyreflective or opaque and may have composite properties, regions withdifferent properties, and may be a composite of more than one material.

Reagents for conducting assays may be stored on the cassette and/or in aseparate container. Reagents may stored in a dry and/or wet state. Inone embodiment, dry reagents in the cassette are rehydrated by theaddition of a test sample. Reagents for conducting ECL assays includeECL coreactants (e.g. TPA), buffers, preservatives, additives,excipients, carbohydrates, proteins, detergents, polymers, salts,biomolecules, inorganic compounds, lipids, and the like. In a differentembodiment, the reagents are stored in solution in “blister packs” whichare burst open due to pressure from a movable roller or piston. Thecassettes may contain a waste compartment or sponge for the storage ofliquid waste after completion of the assay. In one embodiment, thecassette includes a device for preparation of the biological sample tobe tested. A filter may be included for removing cells from blood. Inanother example, the cassette may include a device such as a precisioncapillary for the metering of sample.

The plurality of binding domains and the plurality ofelectrodes/counterelectrodes on the supports are typically placed inregistered proximity to one another by mechanical means, e.g., by usingguide posts, alignment pins, hinges (between each support) or guideedges. Optical guide means may be used to position both supports andelectronic means utilizing optical guide marks defined on the supports.Other systems using electrical or magnetic registration means are alsoavailable.

The supports of the cassette may be configured so as to protect theelectrode pairs from contact with the sample until required to triggeran ECL reaction. For example, the electrodes may be kept separate from abinding domain surface until electrode contact with the sample isrequired by using various mechanical means such as a removable electrodeprotective means.

A cassette or apparatus of the invention comprises reference electrodes,e.g., Ag/AgCl or a saturated calomel electrode (SCE).

The supports may be held together by clips, adhesive, rivets, pins orany other suitable mechanical attachment. They may also be held togetherby the surface tension of a liquid sample or by a compression meansremovably placed on opposite sides of the two supports.

The cassette may also comprise more than two supports, with, forexample, alternating layers of binding domains and electrodes ormultiple supports comprising both a binding surface and an electrodesurface on a single support. This will form a three dimensional array ofECL analysis cells. All of the foregoing components of the cassette aretransparent, except, optionally, some areas between the binding domains.For example, multiple transparent binding surfaces, electrode surfaces,and supports may be stacked.

The first and second supports may be flat and opposed to define asample-holding volume therebetween. Alternatively, the first and secondsupport layers may be configured in other suitable shapes includingspheroidal, cuboidal, cylindrical, provided that the two supports, andany other components thereof, conform in shape. For example, FIG. 10shows a cassette 1100 formed from two adjacent non-planar supports 1102and 1104. Each support has a surface complementary to the other inconformation. Either support may have a PMAMS surface or a multielectrode array or both. One or both of the supports may be elastomericso as to conform to the shape of the other support. The supports or thecassettes may also be prepared in a precut format, or dispensed in asuitable length from a roll dispenser. The cassette may further includesample receiving means such as a sample-holding volume and sampledistribution grooves, channels, indentations and the like.

FIG. 37 shows a cassette where binding domains (3702) in and/or on amatrix (3703) are presented on a surface (3701). A second surface (3700)supporting a working electrode (3704) and a counter electrode (3705) isplaced so that the binding domains are in close proximity to the workingelectrode. Under conditions which lead to light generation from ECLlabel bound to the binding domains, light may be detected through eitheror both surfaces. An array of light detectors (3706, e.g., a CCD array,an intensified CCD array, or an avalanche photodiode array) is used tosimultaneously measure the plurality of light signals from each of thebinding domains. The light detector array images the light generatedfrom binding domains. Lenses, reflectors and/or optical guides may beutilized to enhance imaging. In other examples, light detected fromzones or regions of light detectors (e.g., a light detecting pixel(s))is correlated to a binding domain(s). Image analysis may be used to aidin the correlation of detected light with binding domains. In onefavored embodiment, the surface is elastomeric or compliant andtherefore capable of making intimate contact with the electrodesurfaces. The binding domains are linked to polymers capable of carryingionic currents from the counter electrode to the working electrode. In amore favored embodiment, the objects are water-swollen polymers capableof carrying an ionic current from the counter electrode to the workingelectrode.

FIG. 38 shows a cassette where binding domains (3805, 3806, 3807) arepresented on the surfaces of distinct objects (3808, 3809, 3810)supported on the counter electrode (3800). A working electrode (3801) isplaced in proximity to the surface of the objects. Under conditionswhich lead to ECL from TAGged groups bound to the binding domains, lightmay be detected through either or both of the electrodes (if one or bothof the electrodes is transparent or semi-transparent) and/or from theside. An array of light detectors (3802) is used to simultaneouslymeasure the plurality of light signals from each of the binding domains.The objects may be elastomeric and/or compliant and are thereforecapable of forming intimate contact with the working electrode. Theobjects may be polymers capable of carrying ionic currents from thecounter electrode to the working electrode. The objects may bewater-swollen polymers capable of carrying an ionic current from thecounter electrode to the working electrode.

A transparent support containing one or more binding domains is broughtinto contact with a carbon electrode, e.g. a fibril mat electrode or anelectrode comprised of carbon black or carbon felt. Reagents may beflowed either between the support/binding domains and the fibril mat, orthrough the mat to the binding domains. Light may pass from the bindingdomains, through the transparent support to a detector.

In another preferred embodiment, an electrode is coated with anoptically translucent or transparent layer of carbon (e.g. fibrils) soas to increase the effective surface area of the electrode.

Advantageously, the PMAMS supports and/or cassettes of the invention maybe packaged as kits. The kit comprises one or more PMAMS supportsprepared according to the invention for conducting ECL reactionsincluding assays, controls and the like. Reagents may be optionallyincluded in the kit, including control reagents, ECL assay andcalibration reagents and the like. A reagent mixture may be includedwhich contains a plurality of binding reagents specific for a pluralityof different analytes.

5.9. Apparatus for Conducting ECL Reactions

In one embodiment, the PMAMS on supports, and cassettes containing thesame, are designed to be inserted into an apparatus, that contains meansfor applying one or more test samples onto the PMAMS binding domains andinitiating a plurality of ECL reactions. Such apparatus may be derivedfrom conventional apparatus suitably modified according to the inventionto conduct a plurality of ECL assays based on a support or cassette. Theinvention provides various apparatus adapted to carry out ECL assaysusing each of the specific embodiments of PMAMS described in theSections hereinabove. An apparatus for conducting ECL reactions isdisclosed by Zoski et al. (U.S. Pat. No. 5,061,445). Modificationsrequired include the provision for support and/or cassette handling,multiple sample delivery, multiple electrode addressing by a source fora voltage waveform and multiple ECL signal acquisition and processing.

Elements of illustrative apparatus in accordance with the invention areshown in FIG. 6A. Such apparatus 700 comprises upper and lower supports702, 704 and an electrode guard 710. The upper support bears a pluralityof electrode/counterelectrode pairs (not illustrated). The lower supportbears the binding domains 706. The apparatus is capable of removing theelectrode guard from the cassette and positioning theelectrode/counterelectrodes to contact the analyte bound to the bindingdomains. A reagent or fluid flow space 708 is adjacent to the supportbearing the binding domains. The apparatus is also capable ofsimultaneously or sequentially sending an identical or individuallydetermined voltage wave to each of the plurality ofelectrode/counterelectrode pairs to trigger ECL reactions in thecassette and then measuring the emitted ECL radiation, by a photondetector, e.g., light detector means. The apparatus can further comprisetemperature control means for maintaining the temperature of the supportand/or cassette, or the environment thereon and adjusting thetemperature as needed to optimize ECL reaction conditions. Temperaturecontrol means are preferably heating and cooling means, e.g., electricalresistive heating elements, cooling fans, refrigeration means, and anyother suitable source of heating or cooling. Temperature control meansalso includes temperature sensors, e.g., a thermostat or thermocoupledevice, and means to turn the heating or cooling means on or off inresponse to detected temperature changes.

The apparatus also provides means to hold, move and manipulate one ormore supports or cassettes to conduct ECL reactions. The apparatus mayfurther comprise a stationary or moveable sample delivery means forplacing a sample onto the PMAMS binding domains, as described forcassettes hereinabove.

The apparatus also comprises an electrode contact means able toelectrically connect the array of separately addressable electrodeconnections of the cassette to an electronic voltage waveform generatormeans, e.g., potentiostat (see e.g., FIG. 5 of U.S. Pat. No. 5,068,088).The waveform generator means delivers signals sequentially orsimultaneously to independently trigger a plurality of ECL reactions inthe cassette.

During an ECL assay, ionic current between working and counterelectrodes may flow through ionically conducted liquid (for examplewater containing ionic salts), through a thin film of such liquid,and/or through an ionically conducting solid matrix.

Thus, an apparatus for measuring electrochemiluminescence in a samplecan comprise a plurality of cells for holding at least one sample,wherein a cell may be formed from one or more electrodes and one or morecounterelectrodes and a first support that comprises a plurality ofdiscrete binding domains. The electrodes and counterelectrodes can beprovided on the surface of the first support or on the surface of asecond support wherein the second support is in close proximity to thebinding domains on the first support. The electrodes andcounterelectrodes may occur in pairs. The cell may further comprise aplurality of sensing electrodes to sense the voltage adjacent to theworking electrode. The cassette may further comprise a cell containing areference electrode.

The apparatus further comprises light detection means able to detect ECLreactions conducted in the cassette, e.g., by one or multiple detectormeans. Such detector means include, simply by way of example, an arrayof fiberoptic channels in register with the electrode array andpositioned adjacent thereto, connected to an array of photodetectormeans, or to a single light detector means able to scan the array of ECLsignals as emitted.

The apparatus optionally comprises a digital computer or microprocessorto control the functions of the various components of the apparatus.

The apparatus also comprises signal processing means. In one embodiment,and simply by way of example, the signal processing means comprises adigital computer for transferring, recording, analyzing and/ordisplaying the results of each ECL assay.

Alternatively, the apparatus comprises electrode translation means, forexample, to scan one or more electrode/counterelectrode pairs across thebinding surface to sequentially trigger ECL.

Size exclusion filters may be used in a parallel array of PMAMS.

5.10. ECL Assays That May Be Conducted

ECL labels for use according to the present invention can be selectedfrom among ECL labels known in the art (see Section 2.2, above, and U.S.Pat. No. 5,310,687). The ECL label may comprise, for example, ametal-containing organic compound wherein the metal is selected from thegroup consisting of ruthenium, osmium, rhenium, iridium, rhodium,platinum, palladium, molybdenum, technetium and tungsten. Suitablelinking chemistry for preparing ECL TAG reagents is well known anddisclosed, for example, by Bard et al. (U.S. Pat. Nos. 5,310,687 and5,221,605). The means of attachment of the ECL label to a bindingreagent may be covalent and/or noncovalent. An ECL label may bindnon-covalently to a binding reagent (e.g., through hydrophobic effectsor ionic interactions). In other examples of non covalent attachment,ECL label(s) are bound (covalently or non-covalently) to a complex whichin turn is non-covalently linked to a binding reagent. A more specificexample would be covalent attachment of Ru(bpy)3 through a linker to aNi(II)-trinitrilotriacetic acid complex. This molecule will attach tobinding reagents which include a peptide sequence containing a pluralityof histidines. Other receptor ligand pairs are known in the art whichcan be used in a similar fashion (Sassenfeld, 1990, TIBTECH 8:88-93).Furthermore, an ECL label can be used that contains a multiplicity oforganometallic compounds (e.g., Ru-containing) configured as a branchednetwork (e.g., through a network of hydrocarbon linkers). Such branchednetworks containing a multiplicity of organometallic moieties capable ofECL may be attached once or attached at a plurality of positions on amolecule to be ECL labeled. In another embodiment, the ECL labelcontaining a multiplicity of organometallic compounds is a linearpolymer with the organometallic groups attached at a plurality ofpositions along the length of the polymer chain (e.g., linear, branchedor cyclic polymers).

A plurality of binding domains may be used in a variety of additionalECL assay formats well known to the art. In quantitative assays, a knownamount of ECL labeled reagent is used and the amount of ECL measured iscorrelated to known standards to calculate the amount of analytepresent. Forward, reverse, competitive and sandwich assays can beperformed by methods well known to the skilled worker. In competitiveassays, for example, a method for quantitatively determining the amountof an analyte of interest in a volume of multicomponent, liquid sampleis performed as follows. The binding surface is contacted concurrentlywith (a) a known amount of an ECL labeled ligand that is capable ofcompeting with the analyte of interest in binding to a binding reagentpresent on the binding domains, and (b) sample suspected of containingthe analyte of interest; the contacting being effected under appropriateconditions such that the analyte of interest and the ligandcompetitively bind to the binding reagent. The presence of the analytein the sample will reduce the amount of competing ECL-labeled ligandthat binds to the binding domain, thus reducing (relative to when noanalyte is present in the sample) the resulting amount of ECL. ECL inthe resulting binding domain is triggered and the amount of lightemitted is quantitatively determined, thereby quantitatively determiningthe amount of the analyte of interest present in the sample.Alternatively, the sample may be contacted with the binding surfaceprior to contacting the binding surface with the ECL labeled ligand; theECL labeled ligand will then compete with the previously bound analytefrom the sample on the PMAMS surface and displace some of the previouslybound analyte. In an alternative embodiment, the sample can be treatedso as to contain substances/molecules that are ECL-labeled, and astandard amount of unlabeled analyte of interest can be contacted withthe binding surface prior to or concurrently with contacting of thebinding surface with the sample in order to carry out a competitionassay.

In a sandwich assay, the ECL labeled ligand is a binding partner thatspecifically binds to a second binding moiety on the analyte ofinterest. Thus, when analyte that specifically binds to a bindingreagent in a binding domain of a PMAMS is present in a sample, a“sandwich” is thus formed, consisting of the binding reagent on thebinding domain, bound to analyte from the sample, bound to the ECLlabeled binding partner. In another competitive sandwich assay, copiesof the analyte itself are attached to the binding domains of themulti-array binding surface prior to exposure to the sample. Sample isthen contacted with the binding surface. An ECL labeled binding partner(which can specifically bind to the analyte) will bind the analyte inthe absence of free analyte (from sample) in the assay solution, butwill be competitively inhibited in the presence of free analyte (fromsample) in the assay solution.

In alternative embodiments, sequential labeling is performed. Forexample, in a particular embodiment of a sandwich assay, the analytebound to the binding domain is contacted sequentially with a pluralityof ECL labeled binding partners of the analyte. ECL measurements andoptional washing steps are conducted in between contacting with eachdifferent binding partner. In this way an ECL measurement of a pluralityof different binding moieties of an analyte may be performed (e.g.,CD8⁺, a, b T cell antigen receptor positive T cell). Additionally,multiple ECL labels, each emitting light at a distinguishablewavelength, may each be linked to a different binding reagent specificfor a different moiety on an analyte. Further, a plurality ofdistinguishable reporter means (e.g., ECL label, fluorescent label andenzyme linked label) each attached to a different binding reagentspecific for a different binding moiety of an analyte may be used, forexample, to distinguish a CD4⁺, a, b T cell antigen receptor-positivecell from a CD8⁺ a, b T cell antigen receptor-positive cell.

In preferred embodiments the binding domains contain labelled proteinsand/or nucleic acids and/or cells and/or chemical species. Such labelledcomponents (e.g., ECL labels) may be added to the binding domain duringfabrication, prior to the start of an assay, during an assay and/or atthe end of an assay. For example, multiple labelled components may beadded at various times and sequential readings may be taken. Suchreadings may provide cumulative information. In another embodiment, thebinding domains of the PMAMS may be reused a multiplicity of times.After a given assay is performed, the surface may be washed underconditions which rejuvenates the activity of one or more binding domainsof the PMAMS surface. By way of example, some binding reactions may bereversed by changing the ionic strength of the reaction solution.Alternatively, heat may be used to disassociate binding complexes. Some,binding domains may be inherently self-renewing. Binding domains whichcontain catalytic (e.g., enzymatic) functionalities may be utilized morethan once. The binding domains are utilized continuously, and thus canbe used in biosensor applications.

Additionally, the assay may be formatted so that the binding reagentattached to the multi-array multi-specific patterned surface is ECLlabeled. Upon binding to certain analytes of interest in a sample, theECL signal will be quantitatively modulated. For example, the ECLlabeled binding reagent attached to the surface may be specific for ananalyte on a cell surface e.g., antigens such as alpha and beta T cellantigen receptor antigens, or CD4 or CD8 antigens. Upon exposure to amixture of cells, cells bound to the surface will sterically hinder theability of an electrode surface, brought into proximity with themulti-array multi-specific surface, from exciting the ECL labeledbinding reagent thus down-modulating the ECL signal.

Homogeneous and heterogenous assays may be conducted. In heterogeneousassays, unbound labeled reagent is separated from bound labeled reagent(e.g., by a washing step) prior to exposure of the bound or unboundlabeled reagent to an electrical potential. In homogeneous assays,unbound labeled reagent and bound labeled reagent are exposed to anelectrical potential together. In homogeneous assays, the intensity orspectral characteristics of the signal emitted by the bound labeledreagent is either greater than or less than the intensity of the signalemitted by the unbound labeled reagent. The presence or absence of therespective bound and unbound components can be determined by measuringthe difference in intensity.

Once the desired steps of contacting the binding reagents with analyteor competitor thereof and any binding partners thereto, have beencompleted, one then ensures that the ECL label is subjected to anenvironment conducive to ECL. Suitable ECL assay medium are known in theart. Such an assay medium advantageously includes a molecule thatpromotes ECL of an ECL label, including but not limited to oxalate,NADH, and most preferably tripropylamine. Such a “promoter” molecule canbe provided free in solution, or can be provided by prior linkage to orby production at (e.g., as a product of a chemical reaction) the PMAMSsurface, a monolayer on the surface, the binding domain, the electrodesurface, a binding reagent, and/or an ECL label, etc. If the mediumsurrounding the ECL label bound to the binding domains resulting fromthe contacting steps is conducive to ECL, no changes to the medium needbe made. Alternatively, the medium can be adjusted or replaced toprovide a medium conducive to ECL. An electrode and counterelectrode isalready proximate to the binding domain, or is brought near or incontact with the binding domain, a voltage waveform is applied, and ECLis detected or measured.

In a preferred embodiment of the invention, the above-described steps ofcontacting the binding reagents with analyte or competitor thereof andany binding partners thereto, are carried out in the absence ofelectrodes and counterelectrodes, i.e., such that the sample does notcontact the electrode or counterelectrode. Subsequent to thesecontacting steps, electrodes and counterelectrodes are broughtsufficiently close to the ECL label bound to the binding domain, totrigger an ECL reaction.

A support having a PMAMS may be used for sequencing of nucleic acidstrands. For example, a PMAMS with a plurality of binding domains isprepared with different oligonucleotide probes of known nucleotidesequence as the binding reagents in different binding domains. That is,different binding domains will contain binding reagents of differentknown nucleotide sequence. The oligonucleotide chain or fragments of theoligonucleotide chain to be sequenced are then allowed to bind(hybridize) to the PMAMS binding domains. The nucleic acids to besequenced are ECL labeled. Binding assays are conducted on the PMAMS andthe distribution of ECL signals from the discrete binding domains on thePMAMS is used to sequence the oligonucleotide chain.

The above-described method is based on the ability of shortoligonucleotides to hybridize to their complementary or substantiallycomplementary sequence in another nucleic acid molecule (see, e.g.,Strezoska et al., 1991, Proc. Natl. Acad. Sci. USA 88: 1089-1093; Bains,1992, Bio/Technology 10: 757-58, which are incorporated herein byreference). Conditions can be selected such that the desired degree ofsequence complementarity is necessary for successful hybridization.Hybridization of a DNA molecule of unknown sequence to a probe ofpredetermined sequence detects the presence of the complementarysequence in the DNA molecule. The method is preferably practiced suchthat the hybridization reaction is carried out with the oligonucleotideprobes bound to the binding domains and the sample DNA in solution.

A PMAMS may also be utilized to isolate, screen and/or select a novelmolecule or complex of desired function (e.g. binding or catalysis). APMAMS may be used to isolate compounds and/or lead compounds fortherapeutic uses. A PMAMS containing a plurality of peptides, nucleicacids, viral vectors, or polymers, synthesized by a variety ofcombinatorial chemistries, can be made using the methods of theinvention. A wide variety of such PMAMS treated supports may be used torapidly screen for-binding to, for example, an ECL labeled cellularreceptor. In one method a first PMAMS with a large diversity ofunrelated peptide sequences is used in order to isolate lead bindingpeptide sequences. Then a PMAMS with peptides of related sequences tothose which showed binding to the molecule of interest (e.g., a cellularreceptor) on the first PMAMS are then used. The process is repeateduntil a peptide with the desired binding characteristics are found.

An analyte of interest may be, e.g., a whole cell, a subcellularparticle, virus, prion, viroid, nucleic acid, protein, antigen,lipoprotein, lipopolysaccharide, lipid, glycoprotein, carbohydratemoiety, cellulose derivative, antibody or fragment thereof, peptide,hormone, pharmacological agent, cell or cellular components, organiccompounds, non-biological polymer, synthetic organic molecule,organo-metallic compounds or an inorganic molecule present in thesample.

The sample may be derived from, for example, a solid, emulsion,suspension, liquid or gas. Furthermore, the sample may be derived from,for example, body fluids or tissues, water, food, blood, serum, plasma,urine, feces, tissue, saliva, oils, organic solvents or air. The samplemay comprise a reducing agent or an oxidizing agent.

Assays to detect or measure the following substances may be conducted bythe present invention, by incorporation of a binding reagent specific tosaid substances into the binding domains of the binding surfaces of theinvention: albumin, alkaline phosphatase, alt/SGPT, ammonia, amylase,AST/SGOT, bilirubin-total, blood used nitrogen, calcium, carbon dioxide,chloride, cholesterol-total, creatinine, GGT, glucose, HDL cholesterol,iron, LDH, magnesium, phosphorus, potassium, protein-total, sodium,triglycerides, uric acid, drugs of abuse, hormones, cardiovascularsystem modulators, tumor markers, infectious disease antigens, allergyprovoking antigens, immunoproteins, cytokines anemia/metabolic markers,carbamazepine, digoxin, gentamicin, lithium, phenobarbital, phenytoin,procainamide, quinidine, theophylline, tobramycin, valproic acid,vancomycin, amphetamines, antidepressants, barbiturates,benzodiazepines, cannabinoids, cocaine, LSD, methadone, methaqualone,opiates, pheneylindine, phropoxyphene, ethanol, salicylate,acetaminophen, estradiol, progesterone, testosterone, hCG/bhCG, folliclestimulating hormone, luteinizing hormone, prolactin, thyroid hormonessuch as thyroid stimulating hormone, T4, TUP, total-T3, free-T4,free-T3, cortisol, creatinine kinase-MB, total-creatinine kinase, PT,APTT/PTT, LD ISOs, creatinine kinase ISOs, myoglobin, myo light chain,troponin 1, troponin T, chlamydia, gonorrhea, herpes virus, Lymedisease, Epstein Barr virus, IgE, Rubella-G, Rubella-M, CMV-G, CMV-M,toxo-G, toxo-M, HBsAg (hepatitis B virus surface antigen), HIV 1, HIV 2,anti-HBc, anti-HBs, HCV, anti-HAV IgM, anti-HBc IgM, anti-HAV, HBeAg,anti-HBeAg, TB, prostate specific antigen, CEA, AFP, PAP, CA125, CA15-3,CA19-9, b2-microglobulin, hemoglobin, red blood cells, HBcAb, HTLV, ALT,STS-syphilis, ABO blood type antigens and other blood typing antigens,cytomegalovirus, ferritin, B-12, folate, glycalated hemoglobin,amphetamines, antidepressants and other psychotropic pharmaceuticals.

Measurements of ECL at different binding domains can be donesequentially or simultaneously.

A PMAMS specific for an analyte of interest that is a cell-surfaceprotein is first exposed to a sample containing cells, in which it isdesired to count the cells in the sample. In a preferred embodiment, aknown sample volume and/or diluted sample is exposed to a PMAMS whichhas a multiplicity of binding domains specific for at least one cellsurface antigen. Bound cells can then be quantified by attachment of asecondary binding group linked to an ECL tag. This is a group capable ofinteracting with a broad range of cell types, for example an ECL-TAGlinked to a hydrophobic group capable of inserting into a cell membraneor to a lectin directed against cell surface sugars. The ECL-TAG islinked to a secondary antibody directed against a cell surface antibody.In a more specific embodiment, several cell types bound to the samedomain can be distinguished by the use of multiple ECL-TAG labeledsecondary antibodies. One preferably ensures that the number of discretebinding domains specific for a given analyte on the surface of a cellexceeds the average number of cells that will bind that are present inthe sample. Statistical techniques can then be utilized to determine thenumber of cells per sample volume. This technique can also be used,e.g., to count other particles such as viruses, where the bindingreagent recognizes an antigen on the virus. The domains can be smallcompared to the size of a cell so that only one cell can bind perdomain, thus leading to a digital signal for each domain which can thenbe analyzed over the sum of the domains using statistical methods. Thedomains are large compared to the size of a cell so that multiple cellscan bind to a domain. In this case, the level of signal from each domaincan be calibrated to give the number of cells per volume of sample.Image analysis using an array of light detectors (e.g., a CCD camera oravalanche photodiode array) could be used to count cells and determinecell morphologies.

The invention preferably also provides for methods for conducting ECLreactions, e.g., assays, at a rate of to 1000 ECL reactions in from 5 to15 minutes.

5.11. PMAMS for Use With Other Analytic Methods and/or ECL

The techniques described above for ECL based detection can be used inconjunction with other assay techniques, e.g., as domains in whichcatalyses and other chemical reactions can occur. Discrete bindingdomains according to the invention may be used in other assay techniquessuch as, clinical chemical chemistry assays, e.g., electrolytedeterminations, clinical enzyme determinations, blood proteindeterminations, glucose, urea and creatinine determinations, and thelike. Other assay techniques that may be combined with ECL assays and/orused alone with the PMAMS of the invention include chemiluminescentbased label, fluorescent based assays, enzyme-linked assay systems,electrochemical assays (see, e.g., Hickman et al., 1991, Science252:688-691) and/or resonance detection (e.g., surface plasmon andacoustic techniques) assay systems.

PMAS supports with drops may be utilized in which there is a pluralityof different chemistries within the array of drops. Each drop maycontain different binding reagents and/or different chemical assays(i.e., reaction medium for the same). For example, the drops may behydrophilic, resting on hydrophilic surface binding domains which aresurrounded by hydrophobic surface regions. The drops are protected by ahydrophobic solution covering the surface. The hydrophilic solution tobe assayed is deposited on a second PMAMS with hydrophilic bindingdomains surrounded by hydrophobic regions. The two surfaces are broughtinto registered proximity so as to bring into contact the hydrophilicdomains on the opposite surfaces and a spectral analysis is performed todetect reaction products of the chemical assays.

The fibril mats may be patterned such that there are a plurality ofdiscrete hydrophobic and/or hydrophilic domains surrounded byhydrophilic and/or hydrophobic domains. Drops of aqueous solutionscontaining binding reagents may rest on hydrophilic regions and beconfined by surrounding hydrophobic regions. These drops may contain,for example, fibrils, aggregates of fibrils, binding reagents, ECLreagents, reagents for assays, surfactants, PEGs, detergents, aplurality of biological molecules mentioned above by example, and/orcombinations thereof.

The hydrophobic solution covering the first PMAMS is controllablyremoved (e.g., evaporated, wicked) so as to expose only a portion of thehydrophilic drops at the tops to the environment. A hydrophilic solutionto be assayed for an optical chemical reaction is then exposed to thePMAMS surface—the hydrophilic micro-drops and the solution to be assayedmix and analysis (e.g., spectral) is performed.

PMAMS binding domains may also be used as a pre-filter or filter. Forinstance, a cellular specific PMAMS can be used in certain instancesalone as a filter for certain cell types as well as in conjunction witha size exclusion filter. The resulting analyte solution is then exposedto a PMAMS specific for subcellular particulate matter (e.g., viruses).The particulate subcellular PMAMS and/or a size exclusion filter is usedto generate a small molecule (e.g., protein, small chemical entities)analyte solution. By utilizing a serial PMAMS assay system the analytesolution may be sequentially purified in order to decrease non-specificanalyte interactions.

The optical opacity of a material used for a support, electrode and/orbinding domain may be varied to achieve desired properties. Such amaterial may be translucent, transparent or substantially opaque,depending on the thickness, compositing and/or optical density of thematerial.

The optical opacity of fibril mats increases with increasing thicknessof the mat. Very thin mats are optically translucent. Thicker mats canbe substantially opaque. In some examples, mats that range in thicknessfrom 0.01 μm to 0.5 μm are substantially translucent. In other examples,mats with a thickness greater than 20 μm are substantially opaque. Matswith a thickness between 0.5 μm and 20 μm have intermediate opacity,which increases with increasing thickness of the fibril mat. The opticalopacity of a particular thickness of a mat may depend on thecomposition, density, derivatization, number of layers, types andquantities of materials dispersed in the mat, and/or a combinationthereof. It may also depend on the wavelength of the light used.

If a material is substantially translucent at a given thickness andsubstantially opaque for another thickness, light emitted from a certaindepth in the material may pass out of the material while light emittedfrom another (e.g. greater) depth may be substantially absorbed orscattered by the material. In one example, the variable opacity of amaterial allows the material to be used as an optical filter.

Light emitted from a certain depth in a fibril mat may passsubstantially through the mat and be observed with a detector placed onor in proximity to a surface of the fibril mat. Light emitted fromanother depth may be substantially absorbed and/or scattered by the matand not be observed by a detector placed on or in proximity to thesurface of the mat. This property of a fibril mat (and/or opticallysimilar materials) may be used to distinguish between bound and unboundreagents in ECL assay.

Certain reagents can diffuse (actively or passively), be pulled (e.g.,by suction filtration and/or by capillary action), wicked, or pushed bypressure to a sufficient depth in a porous material that emission oflight from these reagents is substantially or entirely absorbed orscattered by the mat. In one example, a fibril mat acts as both aphysical and an optical filter though which certain reagents are passed,certain reagents are entrained, and/or certain reagents bind to a verythin layer either at or in proximity to the surface of the mat. Reagentbound to one or more binding domains and/or species bound to speciesbound to one or more binding domains (these domains being located eitheron the surface of the fibril mat or in a very thin layer near thesurface of the mat on the PMAMS) are prevented from diffusing, beingpulled, etc. into or through the mat. Reagents and/or other solutionsare flowed or suspended on and/or over the surface of the fibril matsuch that reagents bind only to a very thin layer on the surface of themat. Reagents can be washed through the mats, once or many times, in oneor more directions. Reagents may bind to the fibril mat, one or morebinding domains, other or the same reagents bound to one or more bindingdomains, be entrained inside the mat, pass through the mat, or acombination thereof.

Porous materials used in supports and/or electrodes may have more thanone layer in which the upper layer has binding domains and other layerswithin the mat do not have binding domains. In one example, a fibrilmat, (illustrated schematically in FIG. 29), the upper layer 2900 isthick enough to prevent passage of light that originates in layer(s)2901, 2902 from the mat below this layer. Light 2903 that originatesfrom sources 2904,2905 bound to this upper layer can be detected by adetector 2906 located at or in proximity to the surface of the mat.Light originating from sources 2907, 2908, 2909 in lower layers 2901,2902 is absorbed and/or scattered by either or all layers and cannot bedetected by the detectors 2906, 2910.

A pre-filtration step may be used to select particular sizes, types,derivatives of fibrils and/or fibril aggregates before the mat isfabricated. The filter agent used to filter a suspension of fibrils is amat of fibrils of a certain or many porosities.

A porous material (e.g. a fibril mat) may act as the support for thebinding domains, an electrode which may be used for ECL or otherelectrochemical applications, a filter that can be used to controldelivery of reagents, and/or an optical filter that can transmit, absorband/or scatter light to varying degrees.

5.12. Electrochromic ECL Display Panels

The invention also provides for the production of isolatedelectrochemical pixels for use in flat panel displays. Lithographictechniques have been proposed for use in electrochromic andelectrochemiluminescence based flat panel displays to create pixelswhich when electronically addressed have limited effect on neighboringpixels (i.e., limited cross-talk) (see U.S. Pat. No. 5,189,549). Alimitation of the lithographic technique for reducing such cross-talk isthat the electrolyte material must be capable of changing itsconductivity upon exposure to light. It is a feature of the currentinvention to reduce cross-talk between pixels without the necessity ofusing materials capable of photo-induced conductivity modulation therebyallowing the use of a wide range of different solutions, gels or films.

The two electrode surfaces which are the active region of the pixel areon two surfaces facing each other in a sandwich configuration. Theelectrode surfaces are coated with, for example, complementaryelectrochromic materials. To reduce cross-talk a conductive electrolyticfilm is placed between the electrode surfaces with non-conductiveregions between different electrode pairs (i.e., between pixelelements). If the coated electrode surfaces are hydrophilic then theareas of the surface around the electrodes are made to be hydrophobic(e.g., by means of stamping or deposition through a mask) andhydrophilic conductive droplets are placed on the electrode on the firstsurface (e.g., by means of a fluidics array) and then the second surfaceis robotically aligned and brought into contact with the first surfaceso that the electrodes are in register. The electrolytic droplets canthus be constrained to the area within one pixel without any conductivematerial between pixels. The electrode pairs of a pixel are side by sidein close proximity on the same surface. If the coated electrode pairsare hydrophilic the area encompassing both electrodes is made to behydrophilic with a hydrophobic ring around the hydrophilic electrodearea (e.g., by means of stamping or deposition through a mask). Thedroplets described in the two embodiments above are stabilized usinghydrophobic solutions. The viscosity of the solutions may be increasedto increase the stability of the droplet arrays. The hydrophilicity andhydrophobicity may be reversed. In other embodiments the droplets maycontain solutions capable of polymerizing to increase the stabilityand/or conductivity (e.g., conducting polymers) of the film between orabove the electrode pairs. Additionally, structural features may beutilized to limit cross-talk between pixels. For example, an elastomericstamp (e.g., poly(dimethylsiloxane)) with ring shaped stamp protrusionfeatures capable of circumscribing side by side electrode pixel pairs ona surface may be used to isolate electrolytic solutions, gels, or filmsbetween pixels. Alternatively, side by side electrode pixel pairs may beplaced in electrically insulating well-like structures on a surface,electrolytic solutions, gels or films placed in the wells above theelectrodes, and the entire surface covered or coated to isolate andcontain the electrolytic components of each pixel.

5.13. PMAMS for Use in Other Chemical Reactions

The PMAMS of the invention can also be used to conduct chemicalreactions not in combination with ECL. For example, all the techniquesand non-ECL assays discussed in Section 5.11 above can be used.

A cassette is provided for detecting or measuring an analyte of interestin a sample, comprising: (a) a first support having a plurality ofdiscrete binding domains on the surface thereof to form at least onebinding surface, at least some of the discrete binding domains being ofdifferent binding specificities than other binding domains, each of theplurality of discrete binding domains being hydrophilic and surroundedby hydrophobic regions, and (b) a second support having a plurality ofhydrophilic domains comprising reaction media suitable for conducting achemical assay thereon to form an assay surface, in which the pluralityof discrete binding domains and the plurality of reaction media iscapable of being brought into contact so that a sample to be analyzedpresent on each binding domain is contacted with a reaction medium todetect or measure an analyte of interest. Alternatively, the bindingdomains can be hydrophobic, and the second support has a plurality ofhydrophobic domains containing reaction medium.

The invention also provides a method for detecting or measuring analytesof interest in a sample, comprising: (a) placing drops of a samplecontaining an analyte to be detected or measured on a plurality ofdiscrete binding domains on a support surface, in which the plurality ofdiscrete binding domains comprises at least one binding domain thatcontains binding reagents that are identical to each other and thatdiffer in specificity from the binding reagents contained within otherbinding domains, each of the discrete binding domains beingcharacterized as either hydrophobic or hydrophilic, with the provisothat the region of the support surface surrounding each binding domainis (i) hydrophobic if the binding domain is hydrophilic, and (ii)hydrophilic if the binding domain is hydrophobic, so as to allow one ormore analytes of interest in the sample to bind to the binding domains,and (b) contacting the drops on the first support with a surface of asecond support having a plurality of discrete hydrophilic domainscomprising reaction media suitable for conducting a chemical assaythereon, and (c) determining the presence of the analytes of interestthat are bound to the binding domain.

Also provided is a method for detecting or measuring analytes ofinterest in a sample, comprising (a) placing drops of a samplecontaining an analyte to be detected or measured on a plurality ofdiscrete binding domains on a support surface in which the plurality ofdiscrete binding domains comprises at least one binding domain thatcontains binding reagents that are identical to each other and thatdiffer in specificity from the binding reagents contained within otherbinding domains, each of the discrete binding domains beingcharacterized as either hydrophobic or hydrophilic, with the provisothat the region of the support surface surrounding each of the bindingdomains is (i) hydrophobic if the binding domain is hydrophilic, and(ii) hydrophilic if the binding domain is hydrophobic, so as to allowone or more analytes of interest in the sample to bind to the bindingdomains, and (b) placing drops of a reaction medium on the drops ofsample; and (c) determining the presence of analytes of interest-thatare bound to the binding domain.

In a particular example of this aspect of the invention, bindingdomains, each of which have incorporated a different enzyme thatutilizes as a substrate a sequential intermediate in a chemical reactionare situated on a PMAMS surface, such that the product of a givenenzymatic reaction, which is the reactant for a subsequent enzyme, flowsto the next enzyme in the reaction pathway. The invention also providesfor bulk immobilization of enzymes on self-assembling monolayers, e.g.,for industrial application, using methods as described above.

For example, sheets with such immobilized enzymes on one or both sidesmay be stacked to achieve high surface area to solution volume ratios.Alternatively, such immobilized enzymes may be attached to porousmaterials. Additionally, such immobilized enzymes may be on dipsticks,stirring agents, on the walls of tubes or capillaries, or on the wallsof containers such as an incubator chamber.

In an alternative aspect of the invention, non-ECL assays such asdescribed above can be carried out on PMAMS analogs, said PMAMS analogsdiffering from PMAMS as described above in that the PMAMS analogscontain discrete domains for carrying out non-ECL reactions, thediscrete domains not necessarily having incorporated a binding reagentand therefore not necessarily being binding domains. Such PMAMS analogshave discrete domains for carrying out reactions and are prepared so asto inhibit spreading and/or diffusion of fluid applied to the discretedomains. In one embodiment, the domains are either hydrophobic orhydrophilic relative to the surrounding regions on the support surface,in order to aid in confining the reaction medium and/or sample to thediscrete domains. The use of wells, deposition of reaction medium orsample on felts or porous materials, deposition and drying of reactionmedium or sample on gels, films, etc., can be used to inhibit spreadingor diffusion. Each of such discrete domains is less than 1 mm indiameter or width, preferably in the range of 50 nm to 1 mm, mostpreferably in the range of 1 micron to 1 mm. The same or differentreaction medium can be deposited on each of the discrete domains priorto sample application, or sample application can precede deposition ofreaction medium.

In a preferred aspect of the use of PMAMS analogs to conduct non-ECLassays, drops of reaction medium are placed on a plurality of discretedomains, preferably delivered concurrently from an array of microfluidicguides; and then, optionally, to enhance stability and/or protect thedrop, a more viscous solution (e.g., oil) is placed on top of thereaction medium or, alternatively, in between the discrete domain; andthen sample containing an analyte to be detected or measured is appliedto each domain, either by discrete application to each discrete domainor, in bulk, by exposing the entire surface of the PMAMS analogcontaining the domains to a fluid sample. Any resulting reaction in thebinding domains is allowed to proceed, and the results are observed byuse of a reporter and detection system selected from among those knownin the art.

5.14. ECL Assays Employing the Capture of Particles on Porous Electrodes

The invention includes a method for performing anelectrochemiluminescence binding assay in which a complex is formed. Thecomplex includes, at least, a particle and a label compound capable ofelectrochemiluminescence. The complex may also include ligands used inelectrochemiluminescence assays as disclosed for example in Yang H. J.et al., BioTechnology, 12, (1994), 193-194. The method includes thesteps of (a) forming the complex; (b) collecting the complex byfiltration on a porous, conductive electrode; (c) inducing the labelcompound in the collected complex to luminesce by imposing a voltage onthe electrode; and (d) detecting the emitted luminescence from theelectrode.

In another method for performing an electrochemiluminescence bindingassay, the particle capable of complexing with a component of anelectrochemiluminescence assay is first collected on a porous conductiveelectrode. Then the sample containing the analyte of interest is passedthrough the porous, conductive electrode and forms complex on theparticle theretofore collected on the electrode. Then the label compoundis induced to luminesce by imposing a voltage on the electrode and theemitted luminescence is detected to measure the presence of the analyteof interest. In a preferred embodiment the porous, conductive electrodewill be pre-prepared with particles incorporated therein and upon usethe sample containing analyte of interest will be passed through theelectrode to form the complex.

The invention can be adapted to methods for performing a plurality ofelectrochemiluminescence binding assays for a plurality of analytes ofinterest. In such assays a plurality of complexes are formed, each ofthe complexes including at least a particle and a label compound andthese are collected on a plurality of discrete domains, each of thedomains including a porous conductive electrode. As described, theparticles of the label compound and optionally other assay componentsmay be complexed in solution then collected on the domains or thedomains may contain the particles in the first instance and be complexedwith the label compound and optionally other assay components by passingthe sample through the electrode.

The invention can be adapted for use in standard formats forhigh-throughput assay processing e.g., 96-well on 384-well plates.

In certain preferred embodiments the particles may contain aluminescence species capable of acting as an internal standard in theassay. The luminescence thereof can be measured to calibrate the assay.

The invention includes binding assays in which particles are used assolid-phase supports for binding reagents. The term particle implies norestrictions on the size, shape or composition. The particles arecaptured on a porous electrode by filtration and the presence of analyteis detected by the excitation of ECL from ECL-labels present in thebinding complexes on the particles.

Particle-based assays have been used in ECL assays (see for example PCTpublished applications WO90/05301 and WO92/14139) assays because theyhave a high binding capacity. They also allow the binding event to occurwith kinetic rates approaching those observed for binding events insolution.

Highly sensitive and precise assays have been conducted using a systemthat employs a magnetic field to capture magnetic particles on a metalsurface (see PCT published application WO92/14139; Deaver, D. R., Nature377, (1995) 758-760; Yang, H. J. et al., BioTechnology 12, (1994)193-194). This capture process places the particles in close proximityto an electrode so that excitation of labeled particles can be effected.This technology has been highly successful in many areas. It does,however, have some limitations (primarily cost and complexity) thatrestrict its use in low cost assays employing disposable cartridges.

A system that captures particles by filtration through porous electrodestakes advantage of the high binding capacity and favorable kinetics ofparticle-based assays. It also simplifies the fluidics, may employ alarge variety of inexpensive, non-magnetic, commercially availableparticles, and can use inexpensive, porous, carbon-based electrodes. Itcan also improve the efficiency of ECL excitation of labels bound toparticles. A porous electrode may have a significantly higher effectivesurface area than a non-porous electrode e.g., a metal film. If theelectrode is a fibril mat, which is both porous and comprised of fibrousmaterials, the fibrils may contact, e.g. by wrapping around or layingacross, a substantial fraction of the particle.

The invention includes a cassette containing a porous electrode thatcaptures particles for the detection of analytes by ECL. The cassettemay contain a working electrode comprising a thin ECL-active layer ofcarbon fibrils supported on an ECL-inactive filter (see Sec. 5.1 and thereferences cited therein for a detailed description of carbon fibrils.See Sec. 5.7 for a detailed description of fibril mats). A separatechamber in the cassette contains streptavidin-coated particles and driedbinding reagents, e.g. a biotin-labeled capture reagent and an ECL-taglabeled detection reagent. The cassette also provides a means forintroducing a liquid sample to the chamber containing the particles,means for capturing the particles on the working electrode byfiltration, a counter electrode and a reference electrode. The inventionalso includes associated systems for conducting ECL assays with thecassette, e.g. a housing, electrical connections to the electrodes inthe cassette, a waveform generator or potentiostat, a charge coupleddevice (CCD) for imaging the ECL emitted from the PMAMS, and amicrocomputer for controlling the waveform generator and analyzing theimage received by the camera.

There are several desirable embodiments of the working electrode forparticle-based assays in which the particles are captured by filtration.The material of which the electrode is formed must be capable ofexciting ECL from an ECL label in close proximity to it when anappropriate electrochemical potential is applied. If the electrode isporous, the size of the pores must be large enough to allow filtrationof unbound reagents into or through the electrode but is sufficientlysmall to capture the particles.

Preferably, the working electrode is comprised of a conducting filter.Conducting filters may be formed, for example, from porous carbon,aggregate of particulate carbon, from mats of graphitic fibers, carbonfibrils and/or porous metals that are capable exciting ECL. Theelectrode may be composed of a non-conducting porous material, e.g., acommercially available polymer-based filtration membrane, coated with anECL-active material such as gold, platinum and/or a mat of graphiticfibers). The electrode may have multiple layers. In one embodiment, athin layer of an ECL-active electrode material is deposited on a thickerECL-inactive (but electrically conducting) material. The terms “active”and “inactive” refer to the relative efficacy of the electrode forexciting ECL from an ECL label, this characteristic being dependent onboth the structure of the ECL label and the conditions used to triggerECL in a specific application. The conductive, ECL-inactive layerensures good electrical contact along the entire surface of the activelayer, but prevents the excitation of ECL from unbound ECL-tag labeledreagents that have filtered through the active layer. In a preferredembodiment, the ECL-active layer is a thin mat of carbon fibrils and theECL-inactive support is stainless steel filter paper.

Many techniques for immobilization of binding reagents on particles byeither non-covalent and/or covalent coupling reactions are known in theart. For example, the particles may be coated with streptavidin andspecific binding reagents are then captured using a streptavidin-biotininteraction.

A wide variety of particles suitable for use in the invention arecommercially available. These include beads commonly used in other typesof particle-based assays e.g., magnetic, polypropylene, and latexparticles, particles typically used in solid-phase synthesis e.g.,polystyrene and polyacrylamide particles, and particles typically usedin chromato-graphic applications e.g., silica, alumina, polyacrylamide,polystyrene. The particle may also be a fiber such as a carbon fibril.

Materials are available with a variety of functional groups on theirsurface. This allows for the use of a wide spectrum of immobilizationchemistries. In some assays, the analyte itself may act as a particle.For example, an assay for cells with a specific cell-surface antigen (oran assay to quantify the amount of a cell-surface marker in a populationof cells) may be carried out by treating the cells with an ECL-taglabeled antibody against the antigen followed by filtration of the cellsonto the porous electrode.

This invention may be used to conduct many different binding assaysincluding those described in section 5.10. These include immunoassaysand nucleic acid hybridization assays in both competitive and sandwichformats. Many of these assays detect a labeled analyte or bindingreagent in proximity to an electrode. Conducting the binding reactionson particles in suspension using gentle mixing if necessary isadvantageous because the kinetics of the binding reactions areparticularly favorable (they can approach those of a homogeneousreaction).

Alternatively, the particles can be deposited on an electrode and thebinding reactions carried out by flowing samples past the trappedparticles. Particles bearing binding domains can be deposited on theelectrode in a patterned array, i.e. a PMAMS, by a variety of methodsdisclosed in Sec. 5.1.

In one embodiment, the particles are deposited on an electrode in anarray that corresponds to the pattern of a 96-well plate. Thisparticle/electrode fixture can form the basis of a kit used forhigh-throughput ECL assays. A mask with holes in a 96-well pattern ispressed against the electrode such that the holes in the mask arealigned with the pattern of deposited particles. The walls of the holesdefine the walls of the wells; the electrode and particles define thebottom of the wells and the binding regions. Preferred kits may containany number of holes that meet industry standards, (e.g. 96 or 384 holesfor high throughput screening).

Particles bearing binding reagents may be deposited in a plurality ofzones on an electrode. There may be two or more zones with particlesbearing the same binding reagents. There may be two or more zones withparticles bearing different binding reagents.

A sample may be delivered to one or more zone by fluidic guides or itmay be flushed through all zones in a single step.

Alternatively, the particles may be deposited uniformly on the surfaceof the electrode, (i.e. not in a patterned array) and a fixture withholes may be pressed against the electrode to define the active area ofthe electrode.

It is often desirable to include one or more internal standards.Comparing a signal to one generated by an internal standard cancompensate for variations in the manufacture of an assay cassette or inthe execution of the assay. Dyes may be incorporated into particles asinternal standards. Particles that incorporate fluorescent dyes arecommercially available; some of these dyes can be induced to emit ECL. Adye that differs from a specific ECL-label (for example, by its spectralproperties, by the electrochemical potential at which ECL is emittedfrom it and/or its ECL lifetime) allows for the simultaneous measurementof ECL from the specific ECL label and the internal standard. ECLemissions with different spectral properties can be distinguished by theuse of filters, gratings, and/or other techniques known in the art formeasuring light within a defined spectral window.

Particles can be used to prepare a PMAMS for the simultaneous executionof one or more assays for one or more analytes. By way of example, aplurality of suspensions of particles can be prepared wherein eachsuspension comprises particles bearing immobilized capture reagents. APMAMS is formed by applying microdrops of the suspensions, e.g bymethods disclosed in Sec. 5.1 to defined regions on the workingelectrode.

A cassette may contain only one binding domain for conducting one ECLassay. In this case, the intensity of the ECL may be quantified using asingle light detector. Comparison of the light intensity to thatobtained from known concentrations of the analyte allows forquantification of the analyte. Light detection devices that may be usedinclude photo diodes, photomultiplier tubes and avalanche photo diodes.

The cassette may contain a plurality of binding domains. The emittedlight can be imaged to resolve the signals generated at each bindingdomain. Imaging may be achieved with an array of light detectors such asa CCD camera (see Sec. 5.5). Cross talk between closely packed bindingdomains can be eliminated by positioning an array of lenses over thearray of binding domains. Alternatively, mathematical analysis of theintensities measured at the array of detectors can compensate for suchcross talk.

5.15. ECL Assays Employing PMAMS On Electrodes

The invention includes a cassette containing a PMAMS formed directly onthe surface of an electrode. The cassette contains a working electrodecomprising a thin metal film on a support material. A plurality ofbinding domains, i.e. a PMAMS, are present on the surface of the metalfilm. The cassette also includes a means for introducing fluid samplesand reagents over the surface of the electrode, and a counter electrodeto allow for electrochemical excitation of ECL at the working electrode.A reference electrode may also be included for better control of theelectrochemical potential at the working electrode. An apparatus forconducting ECL assays including a cassette which may comprise a housing,electrical connections to the electrodes in the cassette, a waveformgenerator or potentiostat, a CCD camera for imaging the ECL emitted fromthe PMAMS, and a microcomputer for controlling the waveform generatorand analyzing the image received by the camera.

The formation of PMAMS directly on the working electrode has severaladvantages over previous ECL systems: The combination of the workingelectrode and the solid phase support for the binding assays into oneunit greatly simplifies the manufacture and execution of ECL assays in adisposable format, allowing disposable assays to be produced at lowercost. A plurality of assays can be performed without the use of multipleECL labels. The excitation of ECL from each of a plurality of assays canbe conducted simultaneously by applying a potential to oneworking/counter electrode pair, all of the binding domains being locatedon the surface of the same working electrode). The use of the surface ofa metal as the support for the PMAMS allows the use of well developedtechnology—e.g., the formation and patterning of self-assembledmonolayers (SAMs) on metals—for the formation of the PMAMS.

The working electrode may be made of a wide range of materials includingmetals (e.g., gold and platinum), metal oxide conductors andsemiconductors (e.g., ITO), carbon (e.g., graphite, carbon black, carbonfibrils), and conducting organic polymers (e.g., polythiophene). Theelectrode may comprise a composite of different materials.

In one embodiment, the working electrode is a thin (5 nm -10,000 nm)film on a substrate. The preparation of such films by techniquesincluding evaporation, polymerization, sputtering, chemical vapordeposition, and plating is known in the art. In a preferred embodiment,the working electrode is a thin film of gold evaporated on a substrate.The properties of the substrates for the thin film electrodes can bechosen according to the requirements of the assay system. The substratemay be solid, or if filtration of samples through the electrode orwicking of samples along the electrode is desired, the substrate may bea porous material e.g., a filtration membrane.

Binding reagents may be immobilized by non-specific adsorption directlyto the electrode surface or by covalent attachment to a chemicalfunctional groups on the surface of the electrode. One approach to theintroduction of chemical functional groups on the surface of anelectrode is electrodeposition or electropolymerization of thin films.Another approach is the preparation of self-assembled monolayers (SAMs).Examples of SAMs that can be prepared on electrode materials includemonolayers of organic thiols on gold, and organic silanes on ITO (seeSec. 5.1). As shown in FIG. 50, a SAM may be prepared by the reaction ofa molecule A-L-B 5032 with the surface of the electrode 5033, where A isthe functional group responsible for the attachment of the molecule tothe electrode, L is a linking chain, and B is a functional group whichcan be used for the attachment of binding reagents 5034 to the surface.Alternatively, B may be a binding reagent.

In a preferred embodiment, the SAM is formed by the reaction ofterminally functionalized alkane thiols (HS—(CH₂)_(n)—B) with thin filmsof gold deposited on a substrate. Alkane thiols with a variety offunctional groups (B) can be prepared, allowing for the use of a varietyof immobilization chemistries. For example, if group B includes acarboxylate group, binding reagents which include an amino group can beimmobilized by reaction with the SAM after activation of the SAM withethyl-3-diaminopropylcarbodiimide (EDC) in the presence of(N-hydroxysuccinimide (NHS). Alternatively, functional group B may be amethyl group, binding reagents may be immobilized by non-specifichydrophobic interactions or if functional group B contains a biotinmoiety, streptavidin or other reagents linked to streptavidin eithercovalently or through a biotin-streptavidin interaction) may beimmobilized on the surface.

Many other chemistries for the immobilization of binding reagents areknown in the art and can be employed. In some cases it may be desirableto control the density of functional groups B on the surface of theelectrode in order to control the density of binding reagentsimmobilized on the surface or to maintain some desirable property of thesurface, e.g. resistance to non-specific binding. The control of thesurface density of the functional group B can be achieved by treatingthe electrode surface with a mixture containing the monomers A-L-B andA-L-C in a ratio determined to produce a mixed SAM with the desiredconcentration of B on the surface. The functional group C is chosen tobe resistant to the immobilization chemistry used to couple bindingreagents to B and may have other desirable properties such as producingsurfaces with reduced non-specific binding.

The formation of PMAMS on the surface of an electrode can be achieved bya variety of methods including: (i) photo-lithographic immobilization;(ii) microcontact printing; and (iii) the controlled application ofdrops of binding reagents to the surface through the use ofmicrocapillary arrays or ink-jet printing (see discussion in Sec. 5.1).Patterned SAMs can be used to better define the areas on the surfacewhich are modified with binding domains. For example, microcontactprinting can be used to pattern an area of circles on a gold surfacepresenting a hydrophilic SAM formed from a carboxylic acid terminatedalkane thiol. The remaining gold surface can then be reacted with amethyl terminated alkane thiol to give a hydrophobic SAM. Afteractivation of the surface with EDC in the presence of NHS, drops, eachcontaining a different antibody, are applied to the hydrophilic circles.The drops will be confined to the hydrophobic regions due to thehydrophobic nature of the surface outside the circles, thus allowingcareful control of the area of the immobilized binding domains.

The types of assays which can be conducted using PMAMS immobilized on aelectrode include those described in Sec. 5.10. Many of these assays(for example, immunoassays and nucleic acid hybridization assays in bothcompetitive and sandwich formats) rely on the detection of the bindingto the electrode surface of a binding reagent or analyte that has beenlabeled with an ECL-active group (tag). The intensity of the signalemitted from a tag-labeled reagent on the surface of SAM can be stronglyinfluenced by the nature of the potential waveform used to excite ECL.For example, SAMs of alkanethiolates on gold are good electricalinsulators but highly oxidizing or reducing potentials at the electrodesurface may reduce the insulating properties of the film by introducingdisorder in the monolayer. Excitation of ECL at potentials which do notintroduce disorder into the SAM requires the transfer of electronsthrough the monolayer by tunneling. Much higher intensity signals can beachieved by applying potentials that introduce disorder into the SAM,thus allowing less hindered flow of current to the electrode. Thesepotentials can be applied prior to or during the excitation of ECL.Alternatively, the SAMs could be formed using conditions known in theart to give disordered monolayers. The conductivity of monolayers canalso be increased by including a constituent in the monolayer whichfacilitates the transfer of electrons (for example, by the introductionof a pi-conjugated system into the linking group L). The formation ofSAMs with high conductivity is discussed in more detail in section 5.7.

In some cases the use of potentials which do not introduce disorder intothe SAMs is advantageous. Under these conditions, the discrimination ofbound tag-labeled reagents from unbound tag-labeled reagents in solutionwill be maximized due to the strong dependence of electron tunneling ondistance, thus, eliminating the need for a wash step. An ECL label onthe surface will give a much stronger ECL signal than an ECL label insolution) ECL can be modulated by changes in conductivity between theSAM and the ECL label that result from a binding event. The use of thisapproach to conduct nucleic acid hybridization assays is described inSec. 5.7.

A cassette of the invention may contain only one binding domain forconducting one ECL assay. The intensity of the ECL may be quantifiedusing a single light detector. Comparison of the light intensity to thatobtained from known concentrations of the analyte allows forquantification of the analyte. Light detection devices which may be usedinclude photo diodes, photomultiplier tubes and avalanche photo diodes.A cassette may also contain a plurality of binding domains. The emittedlight from such cassette must be imaged to separate the signalsgenerated at each binding domain. Imaging may be achieved by an array oflight detectors such as a CCD camera (see Sec. 5.5). Cross talk betweenclosely packed binding domains can be eliminated by positioning an arrayof lenses over the array of binding domains or by mathematical analysisof the intensities of the signals from the several binding domains.

5.16. ECL Assays Employing PMAMS On A Porous Substrate

FIG. 37 shows a cassette where binding domains 3702 in and/or on amatrix 3703 are presented on a surface 3701. After completion of bindingreactions on the binding domains, a second surface 3700 supporting aworking electrode 3704 and a counter electrode 3705 is positioned sothat the binding domains are in close proximity to the workingelectrode. Luminescence from an ECL label bound to the binding domainsmay be detected from either or both surfaces. We refer to thisconfiguration for ECL as the “Two Surface” ECL assay.

FIG. 38 shows a cassette where binding domains 3805, 3806, 3807 arepresented on the surfaces of matrices supported on a counter electrode3800. After completion of binding reactions on the binding domains, aworking electrode 3801 is positioned in close proximity to the surfaceof the matrices. Luminescence from an ECL label bound to a bindingdomain may be detected through either or both of the electrodes ifeither or both of the electrodes is transparent or semi-transparentand/or from the side.

The invention also includes an apparatus for conducting ECL assays usingcassettes containing a PMAMS. An apparatus for conducting ECL assaysusing the cassette described in FIG. 38 includes means for makingelectrical connections to the electrodes, means for controlling thepotential at the electrodes, means for moving the matrix into closeproximity with the working electrode and means for imaging the lightemitted during excitation of ECL.

The Two Surface method has several advantages over previous ECL methods.A plurality of assays can be performed conveniently without the use ofmultiple ECL labels. The excitation of ECL from each of a plurality ofassays can be conducted simultaneously by applying a potential to oneworking/counter electrode pair, all the binding domains being placed inproximity to the same working electrode. The working electrode can beprotected during the binding reaction from the sample by a physicalbarrier that is removed prior to the excitation of ECL, thus, preventingcontamination of the electrode surface which could result in a change inits electrochemical performance. As illustrated in FIG. 55 for the caseof a sandwich immunoassay, the binding of ECL-tag labeled reagent 5203to analyte 5202 bound to primary antibody 5201 immobilized on the matrix5200 results in the optimal presentation of the tag 5204 to theelectrode surface 5205, (i.e. with a minimum of organic material—such asprotein, nucleic acid, or linking groups—between the tag and the surfaceof the electrode. The matrix may be used for the concentration and/orseparation of components of a sample, for example, by electrophoresisand/or filtration through the matrix. The matrix may be used as a mediumfor the storage of assay reagents in dried or partially hydrated form.The surface of the matrix can be placed in conformal contact with theworking electrode.

The PMAMS are preferably formed in and/or on a matrix with one or bothof the following characteristics. The matrix is capable of carryingionic currents between the working and counter electrodes, andtherefore, can complete the electrochemical circuit. The matrix ispreferably capable of making intimate contact with the working electrodee.g. it is elastomeric and/or compliant. Materials with thesecharacteristics are known in the art and include porous materials suchas filtration membranes and water-swollen polymeric gels. In someembodiments of the invention, e.g. if light excited at the workingelectrode is detected through the matrix, it is advantageous for thematrix to be transparent.

PMAMS can be generated on porous materials (e.g., gels) with varyingpore size and solvent content. For example, polyacrylamide gels varyingin pore size can be made by varying the concentration of acrylamide andthe degree of crosslinking.

On such matrices with pore sizes smaller than the analyte, bindingreactions will occur substantially on the surface of the gel. In thiscase, filtration and/or electrophoresis through the gel can be used toconcentrate analytes at the surface of the gel and modulate thekinetics, e.g., increase the rate, of the binding reaction. Fasterkinetics are advantageous in rapid assays and may generate increasedsensitivity in a shorter time period.

On matrices with pore sizes larger than the analyte, binding reactionscan occur on the surface as well as the bulk of the gel. In this case,filtration and/or electrophoresis can be used to increase the kineticsof binding as well as to remove unbound species from the surface.

PMAMS formed on gels can be stored wet and/or they may be stored in adried state and reconstituted during the assay. The reagents necessaryfor ECL assays can be incorporated in the gel before storage, bypermeation into the gel or by incorporation during formation of the gel,and/or they can be added during the assay.

The immobilization of binding domains to matrices by covalent andnoncovalent linkages is known in the art. Some examples of methods forthe immobilization of binding domains to a variety of matrix materialsis described in more detail in Sec. 5.1. Sec. 5.1 also describes indetail methods for the patterning of binding domains on a matrix to forma PMAMS. These patterning methods may include the following: i)photolitho-graphic immobilization; ii) patterned application ofmicrodrops of reagents to the surface of a matrix; iii) application ofdrops or microdrops containing the binding domains in the matrix inliquid form to a substrate followed by solidification and/or gelling ofthe liquid using known techniques including, crosslinking,polymerization, cooling below the gelling transition, etc., to givedistinct drops on the substrate composed of the matrix material, eachsolidified drop thereby comprising a different binding domain; iv) usingmatrices to achieve separations, e.g. by electrophoresis in apolyacrylamide slab; and v) Forming a layered structure of matrices eachcontaining one or more binding domains.

The working electrode is preferably made from an electrode material thatis capable of exciting ECL from an ECL label in close proximity to thesurface when the appropriate electrochemical potential is applied. Insome embodiments light is detected from the surface of the PMAMS throughthe working and/or the counter electrode. In these cases, it isadvantageous to use a transparent or semi-transparent electrodematerial. These electrode materials are known in the art. Examplesinclude films made of indium tin oxide as well as very thin (<30 nm)films of gold. Alternatively, it may be advantageous to protect theworking electrode from the sample. A physical barrier on the electrodemay protect it during incubation of the sample with the PMAMS. Thephysical barrier is then removed before placing the PMAMS in closeproximity to the electrode.

5.17. ECL Assays Employing PMAMS On Composite Electrodes

In preferred embodiments of the invention the electrode is a compositeof a polymer containing a multiplicity of carbon fibrils dispersedtherein. Desirably the composite is porous.

A preferred apparatus for conducting an assay comprises as a firstelement a matrix containing carbon fibrils dispersed therein, and one ormore binding domains containing a reagent capable of binding a componentof an assay.

An apparatus for detection of an analyte by electrochemiluminescence maycomprise an electrode comprised of a composite of a matrix having amultiplicity of conducting particles dispersed therein and a bindingdomain containing a reagent capable of binding a component of anelectrochemiluminescence assay. Desirably the matrix is a polymer andthe conducting particles are carbon. The conducting particles aredesirably are carbon fibers and best results obtained were the carbonfibers or carbon fibrils.

Apparatus for use in the detection of a plurality of analytes are alsoincluded in the invention. In such apparatus the electrode is comprisedof a matrix containing a multiplicity of conducting particles dispersedtherein and the plurality of binding domains supported on a surface ofthe electrode, each of those domains containing the reagent capable ofbinding a component of an electrochemiluminescence assay.

The properties of electrodes comprising a polymer and dispersed carbonfibrils may be modified by a subjecting the composite to variouschemical and physical steps such as oxidation, exposure to a plasma andexposure to a reagent capable of derivatizing the electrode by additionof one or more functional groups. In the latter method the polymer canbe derivatized or the fibrils contained therein can be derivatized orboth can be derivatized. Desirably the composite is subjected to achemical or physical treatment to affect the modification for a timesufficient to alter the electrical potential at whichelectrochemiluminescence occurs in an electrochemiluminescent compoundsituated at said composite. It is also within the invention to modifythe properties of any electrode comprising a polymer and multiplicity ofcarbon fibrils dispersed therein by modifying the electrode to expose adesired functional group thereupon. The invention also includeselectrodes which have been modified by chemical or physical treatment toalter the electrical potential at which electrochemiluminescence occurs.

The invention includes a cassette containing a PMAMS formed directly onthe surface of an electrode comprised of more than one material, i.e. acomposite electrode. The several components of such a cassette aredescribed above.

The composite electrode may be comprised of conductive and/orelectrochemically active particles impregnated in a support matrix. Forexample, the matrix may be comprised of oils, waxes, paraffins,plastics, ceramics, teflon, polymers, elastomers, gels and/orcombinations thereof. Some examples of commercial polymers that can beused in the manufacture of composite electrodes include, but are notlimited to, EVA, polyethylene (PE), polystyrene (PS), and ABS.

The matrix can be chosen to meet design requirements for a givenapplication. The material may be appropriate for a specific type ofimmobilization chemistry, it may give high specific signal and/or lowbackground signals in a particular type of assay, and/or because thematerial has desirable physical properties, e.g., flexibility, strength,chemical resistance.

A composite electrode can be formed using any particles that whencombined in a matrix provides an electrically conductive composite. Theparticles may be carbon, e.g. particulate carbon, carbon black, carbonfibers, carbon felts and preferably are carbon fibrils (Secs. 5.1 and5.7).

Composites that contain more than one type of particle and/or more thanone type of material for the matrix can be used. For example, acomposite electrode may include one type of particle to impartelectrical conductivity and ECL-activity and another type of particle asa support for binding domains.

In a preferred embodiment, a composite electrode is comprised of a blendof carbon particles and a matrix. In a particularly preferredembodiment, a composite electrode is comprised of carbon fibrils and apolymer. U.S. Pat. Nos. 5,304,326 and 5,098,771 describe polymercomposites impregnated with fibrils.

Fibril-polymer composite electrodes can be produced by techniques knownin the art of manufacturing plastic materials and parts. For example,flat electrodes can be cut from pressed sheets or extruded films of afibril composite. Electrodes with complicated shapes or surface featuressuch as grooves or channels for the movement of fluid or wells forreaction chambers can be formed by injection molding.

The composite electrode may be a solid or may be porous. Porouscomposites may be formed by using techniques for producing porousplastic materials, e.g., filtration membranes. Filtration through aporous composite electrode can improve the kinetics of binding reactionsto binding domains immobilized on the surface of the electrode.

Binding reagents may be immobilized on unmodified composite electrodes.For example, binding reagents may be immobilized by non-specificadsorption onto the matrix and/or onto the conductive particles.Functional groups present on the matrix and/or the conductive particlecan be used for immobilization of reagents. These reagents can serve asbinding reagents and/or as reagents that change the properties of thesurface e.g. wettability or resistance to non-specific binding. Methodsfor covalent and non-covalent immobilization of reagents on materialsthat can be used as matrices are known in the art (see Sec. 5.1).

In one embodiment, the carbon particles comprise between 0.1% and 99.9%by weight of the composite. In another embodiment the carbon particlescomprise between 0.5% and 50% by weight of the composite. In a preferredembodiment, the carbon particles comprise between 1% and 30% by weightof the composite. In a particularly preferred embodiment, the carbonparticles comprise between 2% and 20% by weight of the composite.

The use of carbon fibrils in composites can be particularlyadvantageous. The conductivity and high aspect ratio of fibrils mayallow preparation of composites that have high conductivity at lowweight-percentage of carbon in the composite (when compared tocomposites containing carbon particles other than fibrils, present atthe same weight-percentage). Composites with low carbon loading thatstill have high conductivity can be advantageous, since high carbonloadings of some carbon particles can compromise the structuralintegrity and/or processability of composites. Composites containingcarbon fibrils can also have high binding capacities due to their largeexterior surface area. Processing of the composite to expose fibrils(e.g. by chemical means or by exposure to a plasma) can alter thebinding capacity, advantageously, to increase it. (By binding capacityhere we mean the amount of a reagent that can be immobilized on a givengeometric area of a material, e.g. nanograms of protein per cm² ofmaterial. By geometric area we mean the area of a material defined bythe dimensions of the material (e.g. a square piece of material withdimensions of 1 cm×1 cm has a geometric area of 1 cm²)). One of thelimitations of many materials is that the binding capacity is limited bythe geometric area of the material. For example, if a smooth, flatsurface is used (e.g. the surface of a metal), it may not be possible toobtain a binding capacity of a reagent (e.g. a protein, nucleic acid, abinding reagent) that exceeds that of a close-packed monolayer of saidreagent distributed over the geometric area of the smooth flat surface.Use of composites with exposed fibrils overcomes this limitation.Exposure of fibrils creates a plurality of protruding fibrils (eachhaving high surface area) at the surface of the composite: the totalsurface area of fibrils available for binding of reagents cansignificantly exceed the geometric area of the composite. In oneembodiment, the binding capacity for a reagent on such a composite canbe greater than 1 times that of a close packed monolayer of said reagentdistributed over the geometric area of said composite. In anotherembodiment, the binding capacity for a reagent on such a composite canbe greater than 2 times that of a close packed monolayer of said reagentdistributed over the geometric area of said composite. In a preferredembodiment, the binding capacity for a reagent on such a composite canbe greater than 10 times that of a close packed monolayer of saidreagent distributed over the geometric area of said composite. Inanother preferred embodiment, the binding capacity for a reagent on sucha composite can be greater than 100 times that of a close packedmonolayer of said reagent distributed over the geometric area of saidcomposite.

In some embodiments, composites containing carbon fibrils can beresistant to damage by certain solvents, temperatures, reagents andprocesses that might otherwise damage the matrix of the composite (ifthe matrix alone were treated). This resistance to damage can beadvantageous in processing. For example, it may be possible to usecertain procedures for derivatization of the composites (e.g. aprocedure that requires a solvent that dissolves the matrix alone, butdoes not appreciably dissolve the matrix or fibrils when they arepresent together as a composite).

The composite electrode may be modified by chemical or mechanicaltreatment to improve the immobilization of binding reagents. The surfacemay be treated to introduce functional groups for immobilization ofreagents. Techniques that may be used include exposure toelectromagnetic radiation, ionizing radiation, plasmas or chemicalreagents such as oxidizing agents, electrophiles, nucleophiles, reducingagents, strong acids,and strong bases and/or combinations thereof (seeSec. 5.18).

One particularly interesting embodiment is the modification of suchcomposite electrode, and more broadly a composite material (not limitedto an electrode) comprising a matrix (such as a polymer) and one or morefibrils and/or fibril structures dispersed therein, by treatment with aplasma. The treatment is carried out in order to alter the surfacecharacteristics of the fibrils, fibril structures and/or the matrix,which come in contact with the plasma during treatment; by this meansthe fibril composite treated can be functionalized or otherwise alteredas desired. Once equipped with the teaching herein, one of ordinaryskill in the art will be able to adapt and utilize well-known plasmatreatment technology (without the need for further invention or undueexperimentation) to the treatment of such composite materials. Thus, thetreatment can be carried out in a suitable reaction vessel at suitablepressures and other conditions and for suitable duration, to generatethe plasma, contact it with the composite material, and effect thedesired kind and degree of modification. Plasmas such as those based onoxygen, ammonia, helium or other chemically active or inert gases can beutilized. Depending on its properties, the modified composition can beutilized as an electrode (such as described above) or for otherapplications.

Examples of other gases used to generate plasmas include argon, water,nitrogen, ethylene, carbon tetrafluoride, sulfurhexafluoride,perfluoroethylene, fluoroform, difluoro-dicholoromethane,bromo-trifluoromethane, chlorotrifluoromethane, and the like. Plasmasmay be generated from a single gas or a mixture or two or more gases. Itmay be advantageous to expose a composite material to more than one typeof plasma. It may also be advantageous to expose a composite material toa plasma multiple times in succession; the conditions used to generatethe plasma, the duration of such successive treatments and the durationof time between such successive treatments can be varied to accomplishcertain alterations in the material. It is also possible to treat thecomposite material (e.g. coat the material with a substance, wash thesurface of the material, etc.) between successive treatments.

Plasma treatment of a composite material may effect several changes. Forexample, a composite material comprising a polymer and a plurality ofcarbon fibrils dispersed therein can be exposed to plasma. Exposure toplasma may etch the polymer and expose carbon fibrils at the surface ofthe composite, thus increasing the surface area of exposed carbonfibrils (e.g. so that the surface area of the exposed fibrils is greaterthan the geometric surface area of the composite). Exposure to plasmamay introduce chemical functional groups on the fibrils or the polymer;these functional groups may be used for the immobilization of reagents.

Plasma may be used to bond reagents to the composite material. Forexample, the composite material may be exposed to a solution containinga reagent (e.g. a detergent, a polyaromatic molecule, a hydrophobicmolecule, a charged molecule, and the like) so that some amount of thereagent coats the composite material. The reagent-coated compositematerial can then be exposed to plasma; exposure to the plasma bonds thereagent to the composite material. In another example, a compositematerial coated with a biomolecule (e.g. a protein, nucleic acid or thelike) is exposed to plasma: exposure to the plasma bonds the biomoleculeto the composite material. In another example, a composite material iscoated with a reagent that binds one or more desired reagentsspecifically (e.g. an affinity chromatography resin, a polymer withbiospecific ligands) and exposed to plasma. Exposure to plasma bondssaid reagent to the composite material.

Reagents bound to composite materials can be used to immobilize otherreagents on the composite. For example, a hydrophobic reagent bonded tocomposite materials may enhance adsorption of proteins to the composite.Affinity chromatography resins, biospecific polymers, proteins and thelike may enhance immobilization of biomolecules to the compositematerials (e.g. biospecifically and/or non-specifically).

Plasma can also be used to induce polymerization of reagents oncomposite materials. The products from plasma induced polymerization canthen be used to immobilize reagents on composite materials. For example,monomeric precursors can be coated on composite materials; exposure toplasma may induce grafting and/or polymerization of some or all of saidmonomeric precursors. In another example, a polymer can be coated on acomposite material by treating the composite with a plasma comprisingthe monomer. In another example, a composite material can be coated withmonomer or polymer by exposing the composite to a plasma so thatpolymerization initiating species are generated on the composite, andthen treating said composite with a monomer.

It may be advantageous to immobilize binding reagents on both the matrixand the particles or it may be advantageous to immobilize bindingreagents on only one of the components, i.e. the matrix or theparticles. By way of example, a composite electrode comprised of fibrilsin EVA (a copolymer or ethylene and vinyl acetate) may be treated with amixture of chromic acid and sulfuric acid to introduce carboxylic acidgroups on the electrode. These carboxylic acid groups can then be usedto immobilize binding reagents containing amines by formation of anamide bond. Alternatively, a composite of fibrils in EVA can be treatedwith sodium hydroxide. In this case, the fibrils remain unmodified buthydroxyl groups are exposed on the polymer. These hydroxide groups canthen be used to immobilize binding reagents containing a nucleophile.

Modification of composites may lead to other favorable properties.Modification of the matrix and/or the particles may produce a compositeelectrode with a high binding capacity. The introduction of hydrophilicgroups to the composite electrode may hydrate the matrix and lead to theformation of a thin water-swollen gel layer. Reagents can be immobilizedwithin such a gel layer, allowing for the immobilization of morereagents than could occupy a flat, solid, surface with the samegeometric surface area. Partial degradation of the matrix can increasethe exposed surface area of the conducting particles and lead to highsurface-area electrodes for the immobilization of binding reagentsdirectly on the conductive particles, especially when the particles arefibers which can extend into the solution.

Modification of a composite surface may shift the electrochemicalpotential required to excite ECL. Modification of the compositeelectrode may reduce or increase the overpotential required forexcitation of ECL from an ECL tag, thereby allowing certain signals,e.g. the signal from an analyte and a background signal, to be resolved.

The formation of PMAMS on the surface of a composite electrode can beachieved by a variety of methods including photolithographicimmobilization, microcontact printing and/or the controlled applicationof drops of binding reagents to the surface through the use ofmicrocapillary arrays or ink-jet printing (see Sec. 5.1). Alternatively,the surface of a composite electrode may be divided into distinctregions by placing it in contact with a mask.

The invention includes a disposable multiwell plate for use in ECLassays (hereon referred to as an “ECL Plate”). In one embodiment, an ECLPlate is manufactured by shaping (e.g., pressing, molding, or forming) aconductive composite into the form of a multiwell microtiter plate. Inanother embodiment, a mask is formed that comprises an array of holesthrough a sheet of a material. Such a mask is then sealed against anelectrode (the electrode is preferably a conducting composite or afibril mat; the preparation of fibril mats is described in detail inSection 5.7, Section 5.18 and the references therein). The holes throughthe mask will then define wells with walls comprising the mask andbottom comprising the electrode. The mask and the electrode may beprovided to the user as a preassembled disposable cassette, or asindividual disposable components of a kit. Alternatively, only theelectrode may be disposable. The electrode may be solid and/or porous.In the case of a porous electrode, binding reactions may be carried outby filtering reagents through the electrode (multiwell filtrationmanifolds for use in binding assays—“dot blots˜—are known in the art).In a different embodiment of the ECL Plate, a plurality of holes in amask (as described in the previous embodiment) is sealed against aplurality of individual electrodes such that the electrodes inindividual wells and/or groups of wells can be individually probed.

An ECL plate is preferable shaped in a standard form used for multiwellmicrotiter plates. These standard formats are known in the art andinclude, but are not limited to, 24, 96, and 384 well plates. The use ofa standard format allows the integration of commercially availableequipment for carrying out binding reactions on microtiter plates (e.g.,equipment for moving plates, washing plates and/or dispensing samples).The invention includes an apparatus for exciting ECL from the electrodeor electrodes of an ECL Plate and quantifying the ECL emitted from eachwell.

ECL plates may be provided to the end user with immobilized bindingreagents for one or more analytes. Alternatively, the user could beprovided with a kit comprising an ECL Plate and the reagents necessaryfor immobilizing binding reagents (when such binding reagents areprovided by the user).

Composite electrodes may be used in assays that do not use ECL. They maybe used as solid-phase binding supports for assays based onfluorescence, chemiluminescence, or ELISA-type formats. They may be usedas electrodes and/or solid phase supports for assays based onamperometric or potentiometric electrochemical detection.

5.18. ECL Assays Employing PMAMS on A Porous Electrode

The electrode of the invention may comprise a mat of a multiplicity ofcarbon fibrils. Such mats have now been found to perform well aselectrodes for use in electrochemiluminescence assays.

The mats broadly comprise a multiplicity of carbon fibrils and at leastone domain containing an assay reagent. In one embodiment of theinvention the mat may be comprised of two or more layers of differentconductivity, two or more layers of derivitized or underivitized carbonfibrils or combinations of derivitized and underivitized fibrils, two ormore layers of fibrils of different optical opacity or two or morelayers of fibrils of different pore sizes.

Desirably these mats are used in electrodes for electrochemiluminescenceassays. The electrode includes a support and a fibril mat comprising amultiplicity of carbon fibrils and means for making electrical contactwith the mat. The electrode may contain a binding domain containing areagent capable of binding a component of an electrochemiluminescenceassay.

The invention includes kits for making electrodes for use in suchassays. The kits include a support, a fibril mat and means for makingelectrical contact with the mat. The fibril mat may include a bindingdomain.

The electrode may be conductive or porous and desirably is conductiveand porous and may be, for example, comprised of a metal-coated porousmaterial. The electrode may be stainless steel fiber mesh.

Fibril mats for use as a support for an electrode in anelectrochemiluminescence assay may be prepared in several differentways. In one such method the fibrils are produced with a binding reagentimmobilized on their surface. These fibrils are dispersed in a medium.They are thereafter filtered from solution to produce a fibril mat.

Alternatively, the fibril mat may be prepared by dispersing the fibrilsin a medium, filtering the fibrils from the medium to prepare the matand finally derivatizing the fibril mat to prepare them for mobilizationof a binding reagent thereupon.

The invention broadly includes methods for performing anelectrochemiluminescence binding assay for an analyte of interest. Themethod includes the steps of (a) an electrode comprised of a conductivepolymer; and (b) a binding domain containing a reagent capable ofbinding a component of a binding electrochemiluminescence assay.

The method of the invention can be used to conductelectrochemiluminescence binding assays for a plurality of analytes ofinterest in a biological sample. This method includes the steps of (a)contacting a sample containing analyte of interest and a label compoundcapable of electrochemiluminescence, with an electrode comprising amultiplicity of carbon fibrils containing a binding domain containing areagent capable of binding a component of an electrochemiluminescenceassay; (b) inducing the label compound at the electrode to luminesce byimposing a voltage thereupon; and (c) detecting the emittedluminescence. Alternatively, the method includes (a) contacting a samplecontaining a plurality of analytes of interest and a label compoundcapable of electrochemiluminescence with a plurality of electrode zones,each of which comprises a fibril mat containing a domain containing areagent capable of binding a component of an electrochemiluminescenceassay; (b) inducing the label compound collected on said fibril mats toelectrochemiluminescence; and (c) measuring the emitted luminescence.

The invention also includes a cassette containing a porous electrode.The cassette contains a working electrode consisting of a porous mat ofcarbon fibrils supported by a porous material. One or more bindingdomains are present on the surface of the working electrode.

The porous electrode may be comprised of carbon e.g., graphitic carbon,glassy carbon or carbon fibers and in a particularly preferredembodiment comprises carbon fibrils. The binding domains of the PMAMSmay be supported by a fibril mat (see Sec. 5.7). The mats may support aplurality of discrete binding domains, any two or more of which may beidentical to each other or all of which may differ from one another. Thefibril mat may, alternatively, support one binding domain.

Carbon fibrils may be prepared with chemical functional groups attachedcovalently or by physical absorption to their surface. These and otherchemical functional groups can be used to attach other materials to thesurface of fibrils. For example, an antibody that can be used in an ECLassay can be attached to one or more fibrils or a fibril mat.

The fibril mat may be in a single layer containing underivatizedfibrils, derivatized fibrils or a mixture of two or more different typesof fibrils. The fibril mat may have two or more layers. Successivefiltration steps may be used to form mats of fibrils composed of one ormore distinct layers that are either in contact with or in closeproximity to one or more other layers. For example, a two-layer fibrilmat may contain a layer of underivatized fibrils and a layer of fibrilsderivatized with a biomolecule. In some multi-layered mats, there may beoverlap or mixing between layers.

The ECL signal that originates from a fibril mat can depend on thecomposition of the mat. For example, different electrochemicalpotentials may be required to elicit ECL at mats of derivatized fibrilsas compared to mats of underivatized fibrils. This differences inelectrochemical potential can be specific for or limited to one or morecomponents of an assay. A mat composed of derivatized fibrils may shiftthe electrochemical potential at which the background ECL signal isobserved to a different electrochemical potential than that at which aECL from a particular species is observed, i.e., the signal from thecomponents of the assay that are desirable to measure, and in doing so,increase resolution between the specific signal and the backgroundsignal.

Different types of fibrils may be used to prepare fibril mats withcertain capabilities. In mats composed of a layer of underivatizedfibrils and a layer of derivatized fibrils, the layer of underivatizedfibrils may provide an electrical connection between an electricalconductor and the layer of derivatized fibrils and also provide physicalstrength. The layer of derivatized fibrils contains one or more of thereactants necessary to conduct an assay. In another embodiment, a matmay contain one or more fibrils derivatized with a molecule that servesas an internal standard for calibration of other signals. A mat may alsocontain a layer of fibrils that are ECL inactive, the inactive layerproviding physical support for a layer of fibrils that are ECL active.

It may be useful to support a fibril mat on another material e.g. on aporous material such as a filter membrane. The fibril mat can be formedon the membrane by filtering a suspension of fibrils through themembrane so as to capture a layer of fibrils on the surface thereof.Multiple-layer mats can be prepared by successive filtering of differenttypes of fibrils.

Electrical connections to fibril mats supported on non-conductingmembranes can be made by contacting one or more electrically conductingelements, e.g. a wire, a metal mesh or a metal ring, with a surface ofthe fibril mat or an electrically conducting element e.g. an array ofmetal pins, can be inserted into or through the fibril mat.

A filter membrane used to support a fibril mat may be electricallyconducting. Examples of conducting filters include metal-coated polymermembranes, conducting polymer membranes, metal meshes, carbon paper,carbon felts, porous metal films, sintered metal filters, metal-fiberfilters and/or metal-fiber papers.

Metal-coated polymer membranes can be prepared by coating these with oneor more metals by thermal evaporation, electron-beam evaporation,sputtering, chemical vapor deposition or plating. In a preferredembodiment, a polymeric filtration membrane is coated with gold bythermal evaporation.

Where filter membranes do not capture fibrils efficiently by filtration,methods can be used to improve filtration efficiency. The effective poresize of the membrane can be reduced by deposition of metals on thesurface and/or interior regions of the filter. The filter membrane canbe partially plugged or occluded with a material of appropriate size,i.e. a filter aid can be used. The filter can be treated chemically toinduce binding between the fibrils and the filter. Binding may be bymeans of covalent bonds, van der Waals forces, hydrogen bonding,charge/charge interactions, or by hydrophobic hydrophilic interactions,or by biospecific bonding (protein/ligand, antibody/antigen, etc.). Thefibrils may be captured by other mechanisms e.g. deposition on thesurface of the filter by evaporation of the liquid in which they aresuspended.

A filter that supports a fibril mat can work as an electrode for ECL.Examples include filters composed of, or coated with, gold, platinum,carbon, and/or indium-tin oxide(ITO). In such embodiments, both thesupport and the fibril mat may contribute to the observed ECL signal. Insome embodiments, a filter that supports a fibril mat does not functionas an electrode for ECL. Such filters provide support and electricalconnectivity for the fibril mat, but do not contribute to the observedECL signal including the background ECL signal.

Fibril mats can also be supported on non-porous materials. Fibril matsmay be supported on a material capable of acting as an ECL electrodesuch as gold foil, platinum foil, conducting composites or ITO. Fibrilmats may be supported on a material that cannot function as an ECLelectrode such as stainless steel, nickel or non-conducting materials.

PMAMS can be prepared on fibril mats. The reagents necessary to form thePMAMs are delivered to spatially distinct regions of a previously formedfibril mat by microfluidic guides as described previously. For example,an array of microfluidic guides (G1, G2, . . . Gn) can be used todeliver biotinylated antibodies (A1, A2 . . . An) to spatially distinctregions of a mat composed of streptavidin-coated fibrils. Derivatizedfibrils may be delivered to spatially distinct regions of a support bymicrofluidic guides where they are captured, e.g. by filtration orevaporation. For example, an array of microfluidic guides (G1, G2, . . .Gn) can be used to deliver fibrils (F1, F1, . . . Fn) covalently linkedto antibodies (A1, A2 . . . An) to spatially distinct regions of agold-coated ultrafiltration membrane. In yet another method, asuspension of fibrils may be filtered through a physical mask, e.g. awire mesh, placed in contact with a filter membrane so that fibrilsdeposit on the filter wherever the filter is exposed by the spacesbetween the wires of the mesh.

The types of assays that can be conducted using PMAMS immobilized onfibril mats include those described in section 5.10. Because the fibrilmat is porous, it is possible to conduct assays by flowing the reagentsthrough the fibril mat and in some cases the underlying support. Becausethe size of the pores in a fibril mat may be small (for example,10-10000 nm), flowing the reagents through the mat mixes the reagentsefficiently. This reduces the time required to conduct an immunoassay byimproving the rate of mass transfer to the surface of the bindingregions. Assays conducted by wicking a sample into or through the matbenefit similarly from increased kinetics. Alternatively, the fibril matmay be soaked in the sample. The fibril mat can also act as a filter toremove unwanted materials from biological samples.

Fibril mat electrodes may be used in assays that do not use ECL. Theymay be used as solid-phase binding supports for assays based onfluorescence, chemiluminescence, or ELISA-type formats. They may be usedas electrodes and/or solid phase supports for assays based onamperometric or potentiometric electrochemical detection.

5.19. Methods For Increasing Signal To Background

It has also now been discovered that two or more signals originatingfrom electrochemiluminescence species in an electrochemiluminescenceassay can be resolved by conducting the assay at an electrode having atleast two zones which have different electrochemical potentials at whichelectrochemiluminescence occurs. By this method it is possible toresolve signal from background electrochemiluminescence and therebysignificantly improve the performance of the assay.

Another method for resolving two or more signals originating fromelectrochemiluminescence species in an assay comprises including in theassay a reagent which selectively modulates the electrochemiluminescenceof one of the electrochemiluminescence species. For example, a reagentcan be included which quenches electrochemiluminescence from one of thespecies.

Another method for resolving two or more signals fromelectrochemiluminescence species comprises conducting the assay and anelectrode which includes one zone which is inactive for generatingelectrochemiluminescence from one or more of the species in the assay.

Background signals can be distinguished from a desired signal in anassay by conducting the assay at an electrode which induceselectrochemiluminescence for the label and for the background,respectively, at different electrochemical potentials. Likewise, thesignals from two or more species labeled with the sameelectrochemiluminescent compound can be distinguished from one anotherat an electrode which induces the electrochemiluminescence from each ofthe labels at different potentials. These improved methods can becarried out on composite electrodes, desirably those comprised of carbonand best results are obtained by those which have been modified bychemical or physical treatment to change the electrochemical potentialat which electrochemiluminescence takes place.

The invention includes methods for performing anelectrochemiluminescence binding assay for an analyte of interest whichcomprises the steps of (a) contacting a sample containing analyte ofinterest and a label compound capable of electrochemiluminescence, withan electrode comprising a multiplicity of carbon fibrils containing abinding domain containing a reagent capable of binding a component ofthe assay, the carbon fibrils having been modified by chemical orphysical treatment to alter the electrochemical potential at whichelectrochemiluminescence of at least one species in the assay occurs;(b) inducing the label compound at said electrode to luminesce byimposing a voltage thereupon; and (c) detecting the emittedluminescence.

The demands of researchers and clinicians make it imperative to lowerdetection limits of assays, to increase the sensitivity of those assaysand to increase the speed at which these assays are performed.

A critical parameter in meeting these demands is the optimization of thesignal to background ratio. Here, the signal to background ratio (S/B)is defined as the ratio of the signal from components of the sample thatare desirable to measure, (e.g. an analyte) to the signal fromcomponents of a sample that are not desirable to measure (e.g.contaminants). Optimization of the S/B ratio generally involvesmaximizing the signal from components that are desirable to measure andminimizing the background signal.

Various methods are known in the art for increasing the signal fromlabeled species. For example, in U.S. Pat. No. 4,652,333 particleslabeled with fluorescent, phosphorescent or atoms fluorescent labels canbe concentrated by microfiltration before a measurement step isperformed.

Various methods are also known in the art for reducing the backgroundsignal. Wash steps have been used to remove contaminants, unboundanalytes, unbound labeled species, or other components of the sample.

It is advantageous to resolve two or more signals such that detection ofone or more signals is optimized.

FIG. 58 illustrates a method in which the electrochemical potential ofthe ECL signal for one or more components is shifted. In FIG. 58A, theECL signals for two components (A and B) of a sample appear at similarelectrochemical potentials. As such, they are difficult to resolve. FIG.58B shows ECL signals for two components (A and B ) that appear atdifferent electrochemical potentials. The potential of the ECL signalfor component B has shifted to a different potential and the ECL signalsfor components A and B are readily resolved.

The selective shift in electrochemical potential that is illustrated inFIG. 58 can be accomplished by choosing a material for a workingelectrode according to the electrochemical potentials at which ECL iselicited from one or more labels in proximity to the electrode.Alternatively, a material may be modified by chemical or mechanicaltreatment so as to change the electrochemical potential of the ECLsignal for one or more components of a sample.

The electrode may have two or more regions with differentelectrochemical properties so that one region of the electrode excitesan ECL label at a different electrochemical potential than anotherregion. The electrode may be a two-layered fibril in which layer 1 hasbeen derivatized with a binding reagent that binds one or more analytesfrom a sample and molecules bearing ECL labels and layer 2, conversely,has not been derivatized. It does not bind the analytes but can interactwith other components of the sample that give a background ECL signal.As a consequence of the derivatization, layer 1 has differentelectrochemical properties than layer 2. The ECL signal from labeledmolecules bound to layer 1 appears at a higher electrochemical potentialthan the background ECL signals that originate from layer 2.

In another embodiment, a compound that changes the electrochemicalproperties, e.g. the electrochemical potential at which ECL is elicitedfrom a label and/or the intensity of the ECL signal of one or morecomponents in a sample, can be added to the sample.

FIG. 59 shows a schematic of another method for resolving two or moresignals. The intensity of the ECL signal for one or more components of asample is reduced relative to the intensity of the ECL signal for othercomponents of the sample. In FIG. 59A, two components (A and B) have ECLsignals that appear at similar electrochemical potentials. In FIG. 59B,the value of the intensity of the ECL signal for component B has becomesmaller relative to the intensity of the ECL signal of component A′.

The selective change in the intensities of ECL signals illustrated inFIG. 59 can be accomplished by adding a material that quenches the ECLsignal for one or more components of a sample. Alternatively, Theworking electrode may have two or more regions with differentelectrochemical properties, e.g., an electrode may have one or moreregions (R1) that can trigger ECL (“ECL-active”) and one or more regions(R2) that cannot trigger ECL (“ECL-inactive”). Components (A1) of asample bound to regions R1 give an ECL signal in the presence of anappropriate electrochemical potential while the components (B2) of asample bound to R2 give no ECL signal.

The term “ECL-inactive” can also describe regions of an electrode thatproduce a non-zero ECL signal that is substantially smaller than the ECLsignal from other regions of an electrode or a different electrode. Agiven material may be ECL-active under some conditions, e.g. in thepresence of buffers or certain ECL labels and be ECL-inactive underdifferent conditions.

An electrode can be composed of fibrils, which are ECL active, and asupport which is ECL inactive. In this embodiment, the components of thesample that are in electrochemical contact with the fibrils emit an ECLsignal when the proper electrochemical potential is applied. Incontrast, components of the sample that are in electrochemical contactwith the ECL-inactive support and not the fibrils do not give an ECLsignal when the electrochemical potential is applied.

The optical opacity of an electrode can be used to selectively preventdetection of ECL signals from one or more components of a sample (seeSec. 5.11 and FIG. 29).

An electrode may be ECL active for one or more components of a sampleand ECL inactive for other components.

In another embodiment, one or more components (A_(n)) of a sample can bein electrochemical contact with an ECL active electrode and one or morecomponents (B_(n)) of a sample can be out of electrochemical contactwith an ECL electrode, i.e. they are not in sufficient proximity to theelectrode. When an appropriate electrochemical potential is applied tothe electrode, an ECL signal originates from components A_(n) and notfrom components B_(n). An electrode may consist of a porous, ECL activelayer bearing one or more binding domains for analytes A_(n) and aporous, ECL inactive layer. When a sample is filtered through thiselectrode, some analytes A_(n) bind to the ECL active layer, and unboundcomponents are captured in the ECL-inactive layer. When a potential isapplied to the electrode, ECL is triggered for the bound components A,since they are bound to an ECL active layer, but not for the othercomponents, since they are entrained in an ECL inactive layer. Theinvention is further described in the following examples which are in noway intended to limit the scope of the invention.

5.20. ECL Assays Employing Sonication

The disclosure of commonly-owned copending U.S. patent application Ser.No. ______ entitled ASSAY SONICATION APPARATUS AND METHODOLOGY filed oneven date herewith is hereby incorporated by reference in its entirety.In many diagnostic systems wherein binding reactions occur betweenreagents, improved mixing of the reagents can increase the speed of thereaction. Often, the slow rate of mixing ultimately limits the speedwith which a diagnostic test proceeds to completion. Examples ofdiagnostic assays wherein binding reactions between reagents occurinclude immunoassays, DNA-probe assays, clinical chemistry tests,receptor-ligand binding assays, and the like. The slow rate of bindingkinetics has been an especially limiting constraint in conducting assaysthat incorporate binding reactions between reagents in solution andreagents present on a solid. Sonication improves the mixing of reagentsin solution and the mass transport of reagents in solution to reagentslocated on or near a surface of a solid. Experiments have proven thatsonication of assay reagents dramatically decreases the time required toconduct a binding assay that utilizes a solid-phase support. Sonicationis defined to encompass vibration having a frequency betweenapproximately 100 Hz and 10 MHz. The frequency of sonication (f_(s)) canbe sub-divided into the following ranges: low-frequency sonication (100Hz≦f_(s) 5 KHz), ultrasonication (between 5 KHz≦f₅ 1 MHz), andultra-high sonication (1 MHz≦f_(s)≦10 MHz). The amplitude of thevibrations can be sub-divided into the following ranges: low amplitudesonication (<1 μm), medium amplitude sonication (1-10 μm) and highamplitude sonication The improved mixing achieved by sonication findsready and useful application in both end-point and kinetic assays. In anend-point assay, the concentration or amount of an analyte of interestis determined by measuring how much binding has occurred when thebinding reaction has approached completion. We have found thatsonication during the course of the binding reaction decreases the timerequired for the binding reaction to approach completion. In a kineticassay, the concentration or amount of an analyte of interest isdetermined by measuring the rate of the binding reaction. Similarly, ithas been found that sonication during the course of the binding reactionincreases the rate of the binding reaction. The faster binding reactionproduces measurable signals in much less time than previously possible.The invention so greatly accelerates the rates of certain reactions thatassays utilizing such reaction may be completed in only a matter ofminutes, often in less than three minutes.

The rate of a mass transport-limited binding reaction on a solid supportmay be a function of both the concentration of the soluble reagent andthe mass-transport coefficient for the mass-transfer of that reagent tothe solid support. Therefore, it is especially important that theamount, rate, and type of sonication applied during a kinetic assay becarefully controlled and be precisely reproducible. Variations in themass-transfer coefficients are likely to cause variations in reactionrate among otherwise identical tests and, consequently, render impreciseor entirely unusable results. The use of a sonication devicestructurally coupled to an assay cell and/or to a solid-phase supportenables the conduct of kinetic binding assays that are quick,quantitative, highly sensitive, and reproducible.

It has been found that ECL sandwich immunoassays using captureantibodies located on a working electrode, the binding reaction can takemore than ½ hour to reach completion, even when vortexing is used toincrease mass transport to the solid-support surface. This time scale isalso typical of other highly sensitive solid-phase binding assays, suchas ELISA and RIA. Unexpectedly, we found that sonication of reagentsreduced the time required for completion of these binding reactions to amatter of minutes. The apparatus and methodology of the invention is notlimited to immunoassays and will be useful for a wide variety of bindinginteractions (e.g., nucleic acid hybridization, antigen-antibody,receptor-ligand, enzyme-substrate, etc.).

Sonication is also advantageously employed in systems where thesolid-phase support has a plurality of binding domains, and each of saidbinding domains reside on a different location on the solid phasesupport. In this case obtaining accurate and reproducible resultsrequires that the sample be mixed sufficiently so that all portions ofthe sample are exposed to all binding domains. Sonication, by makingmass transport efficient, enables this process.

It is also advantageously employed in systems where the solid-phasesupport has a plurality of binding domains, some or all of said findingdomains being specific for a different analyte. Obtaining accurate andreproducible results requires that all portions of the sample be exposedto all binding domains on the support (e.g. if a certain portion of thesample was not properly mixed, and therefore was not exposed to a regionof the binding surface that had a binding domain specific for theanalyte contained in said portion of the sample, a false “negative”result could be obtained.

Apparatus according to the invention provide a more-than three-foldincrease in the ECL signal produced by a solution containing TAG1 andthe ECL coreactant tripropylamine (TPA) when the experimental cell issonicated during the excitation of ECL. The present invention can,therefore, be applied to the more sensitive ECL detection of ECL labelsand ECL coreactants.

Sonication will not only increase the rate of mass transport of reagentsto a surface of a solid but will also increase the rate of masstransport of reagents, products, byproducts, contaminants, and the likeaway from the surface. Sonication can be used to increase the rate ofdisplacement reactions, e.g., the displacement by an unlabeled analytepresent in a sample of a labeled analyte bound to a binding reagent.Sonication may also be used to increase the rate of desorption ofundesired contaminants on a solid-phase support, thus, reducing theamount of interference and non-specific binding produced in a particularassay. Further, sonication may increase the rate of adsorption ofdesired materials, such as a protective coating, or the like, andincrease the rate of desorption of expended or otherwise undesirablematerials, such as a protective coating, or the like. Sonication may beused to re-suspend particulate contamination, e.g., cell membranes orparticulate reagents, that has settled on a surface.

Sonication may also be used in a sample preparation step. For example,sonication may be used to disrupt materials such as biological tissuecells, microorganisms, virus particles and the like, to releasecomponents of the materials into the reaction media. Preferably, saidsample preparation occurs, in situ, in a measurement cell, e.g., an ECLcell.

Still further, sonication may be used to decrease the time needed to mixtwo or more solutions to homogeneity, the time needed to dissolve asolid in a solution, and the time needed to rehydrate a dried material.Sonication is also useful in increasing the rate of fluid flow throughthin capillaries.

Sonication may be created by a variety of mechanical andelectromechanical devices. Such devices include electric motors with aneccentrically mounted cam, electromagnetic speakers, crystaloscillators, pendulum devices, and the like. A preferred device forcreating sonication at a frequency and amplitude particularly suitablefor the present invention incorporates a piezoelectric material.Piezoelectric materials are generally inexpensive, commonly available,lightweight, and can be induced to sonicate over a wide range offrequencies and amplitudes. Conveniently, piezoelectric sonicationdevices are usually rather small in size, making them especially usefulin desktop and portable devices. Most advantageously, piezoelectricdevices may be operated with very small amounts of electrical power.Sonication apparatus according to the present invention are effectivelysonicated with piezoelectric devices that consume less than ten watts,and a particular apparatus functions with a piezoelectric deviceconsuming approximately 0.25 watts. A preferred piezoelectric device isa piston-mass device.

It was further discovered that structural coupling of sonicating energyfrom a sonication generator to a cell containing assay materials is aremarkably efficient design. The most effective structural coupling hasproven to be solid contact, e.g. by direct attachment of the sonicationgenerator to the cell or attachment of the sonication generator so thata solid continuum is provided between the sonication generator and theassay cell. By specifically transmitting sonication energy to the assaycell or to a solid-phase support in the assay cell, much less energy isneeded as compared to inducing an entire apparatus to sonicate. Carefulpositioning of the sonication generator allows focused direction of theenergy of the contents of the assay cell and lessens the effects ofdamping by other elements of an assay system. Structural coupling may bereversible (e.g. the sonication generator and the cell may be designedto be connected and unconnected multiple times) or may represent apermanent connection.

It is to be understood that structural coupling of sonication energy canbe achieved with many different types of configurations. The structuralcoupling of sonication energy specifically encompasses the transmissionof sonication energy (a) through a solid interface between a sonicationgenerator and an assay medium or binding surface; or (b) from asonication generator directly to an assay medium or to a bindingsurface.

It is an important advantage of the invention that the structuralcoupling of sonication energy in apparatus according to the presentinvention can be precisely controlled. Such control of the structuralcoupling mechanism is readily implemented through precise control of themanufacturing apparatus components and the assembly of same. Since eachcomponent of the structural coupling mechanism, e.g. the sonicationgenerator, the diaphragm, etc., can be composed of rigid materials, eachcomponent can be manufactured to precise tolerances. Similarly, thestructural coupling mechanism is suitable for precise, rigid assemblypermitting the construction of multiple apparatuses having virtuallyidentical sonication transmission characteristics.

The present invention is generally applicable to binding assay systemssuch as immunoassays, nucleic acid hybridization assays, receptor-ligandbinding assays, and the like. In assays where binding reactions occur inthe vicinity of an electrode, sonication of the electrode itself hasproven to have an especially beneficial effect in increasing assayreaction rates.

FIG. 68 illustrates a particular cross-sectional view of an assay cell68010 according to an embodiment of the present invention. Assay cell68010 comprises a base 68011, a diaphragm 68013, and a sonicationgenerator 68016. Base 68011 is shaped to define a cavity 68017 and anaperture 68014, and is preferably a rigid material. Alternatively, base68011 comprises a flexible material (e.g., base 68011 comprises aflexible plastic container or a blister pack). In assay formats that useoptical detection techniques (e.g., ECL, fluorescence,chemiluminescence), base 68011 is preferably a transparent material,such as acrylic or the like, that allows light generated within cavity68017 to be detected by a detector (not shown) coupled to base 68011.

Diaphragm 68013 is a solid-phase support for a reagent 68015, such as abinding reagent, and preferably is comprised of a thin film or sheet ofmaterial. In particular, diaphragm 68013 is preferably a fibril-polymercomposite material. As shown, diaphragm 68013 is coupled to base 68011at aperture 68014. Preferably, diaphragm 68013 forms a seal with base68011 covering aperture 68014.

Sonication generator 68016 is a device for sonicating diaphragm 68013.Preferably, sonication generator 68016 comprises a piezoelectricsonication device. Generator 68016 is preferably controlled by asonication generator controller (not shown) such as an electricalcontrol circuit or the like. Sonication generator 68016 is structurallycoupled to diaphragm 68013 so as to efficiently transmit sonic energy todiaphragm 68013 and to reagents 68012.

In operation, reagents 68012 are introduced into cavity 68017.Sonication generator 68016 is energized and sonicates diaphragm 68013.Diaphragm 68013 conducts the sonication energy to cavity 68017, and thusto reagents 68012 contained therein. The sonication causes reagents68012 to mix, speeding the rate of reaction among reagents 68012. Thesonication will also increase the rate of mass-transport of reagents,products, byproducts, etc., to and from binding reagents 68015 ondiaphragm 68013, thus, speeding the rate of binding reactions at thesolid-phase support. Alternately, binding reagents 68015 may be omitted.

In an alternate embodiment, a non-solid coupling material (not shown) isplaced between generator 68016 and diaphragm 68013. The couplingmaterial may be liquid or gas. It is contemplated that the couplingmaterial may be held in a sealed container, such as a flexible plasticmembrane. In another embodiment, the coupling material may comprise asolid piston structure. Sonication energy from sonication generator68016 is structurally coupled via the solid piston structure todiaphragm 68013. In a further alternate embodiment, reagent 68015 isomitted from the surface of diaphragm and is located on a surface ofcavity 68017.

An assay system 690100 for conducting ECL assays in a disposablecartridge 69090 with an instrument 690101 is illustrated in FIG. 69.Cartridge 69090 includes a base 69091, a diaphragm 69092, acounterelectrode 69093, a reaction enclosure 69094, a sample port 69095,electrical leads 69096, and a reference electrode 69099. Instrument690100 includes a cartridge receptacle 690108, a light detector and/orimaging device 690102, an electrical connector 690103, a source ofelectrical energy for applying a voltage or current between the workingand counter electrodes 690104; a sonication device 690105; a source ofelectrical energy 690106 for driving sonication device 690105; and amicroprocessor 690107 for instrument control, assay data gathering, andassay data analysis.

Diaphragm 69092 is an electrically conductive solid-phase support forreagents 69097A, such as binding reagents, and functions as a workingelectrode. In a preferred embodiment, diaphragm 69092 is afibril-polymer composite electrode and reagents 69097A comprise bindingreagents such as antibodies, nucleic acids, receptors, etc. immobilizedthereon. In an especially preferred embodiment, binding reagentsspecific for a variety of analytes are patterned into binding domains ondiaphragm 69092. Base 69091 is preferably a rigid and transparentmaterial, such as acrylic or the like, that allows light generated by anECL reaction occurring within enclosure 69094 to be detected by detector690102. Base 69091 is shaped to define reaction enclosure 69094 andsample port 69095. Diaphragm 69092 is preferably sealed to base 69091.

Electrical leads 69096 are electrical contacts providing electricalcoupling to diaphragm 69092 and to counter electrode 69093. Preferably,diaphragm 69092 is mounted such that the transmission of sonicationenergy from device 690105 to base 69091 is minimized. Alternatively,diaphragm 69092 may be mounted so that diaphragm 69092 transmitssonication energy from device 690105 to base 69091, and thereon to theentire surface of reaction enclosure 69094.

Preferably, reaction enclosure 69094 is partially defined by the innersurface of base 69091. Alternatively, reaction enclosure 69094 maycomprise a separate enclosure made of a transparent material whichcouples to base 69091.

Counter electrode 69093 is preferably an electrically conductivematerial, such as metal. Reference electrode 69099 is preferably anAg/AgCl reference electrode. Electrodes 69093 and 69099 are disposedwithin base 69091, are coupled to leads 69096, and are adapted to be inelectrical contact with reagents 69098. Optionally, reference electrode69098 may be omitted. Aperture 69095 is preferably adapted for insertionof sample material (e.g., reagents 69098) via a small tube (not shown),such as a capillary tube.

The inner surface of instrument 690101 is adapted to receive and aligncartridge 69090 and its components with receptacle 690108 and itscounterpart components, including sonication device 690105, electricalconnections 690103 and detector 690102. Preferably, detector 690102 isan array of detectors (e.g., a CCD camera or a photodiode array) thatcan image the light emitted during an ECL reaction at the workingelectrode. Detector 690102 may be a single detector such as aphotomultiplier tube, a photodiode, or the like. Insertion of cartridge69090 in instrument 690101 aligns detector 690102 with base 69091 suchthat detector 690102 is positioned to detect much of the light producedwithin enclosure 69094.

Sonication device 690105 is a device for sonicating diaphragm 69092which transmits the sonication energy to reagents 69098 contained inreaction enclosure 69094. Insertion of cartridge 69090 in instrument690101 preferably aligns device 690105 with the center of diaphragm69092 such that device 690105 may be moved into contact with diaphragm69092. Insertion of cartridge 69090 in instrument 690101 causessonication device 690105 to be structurally coupled to electrode 69092.It is preferred that sonication device 690105 comprises a piezoelectricsonication device that may include a piston. Preferably, sonicationdevice 690105 is movable to achieve contact with diaphragm 69092 whencartridge 69090 is inserted into instrument 690101.

Upon insertion of cartridge 69090 into receptacle 690108, electricalleads 69096 are coupled to electrical connections 690103. The source ofelectrical energy 690104 may be a controllable voltage or current sourceadapted for control by microprocessor 690107. Alternatively, ifcartridge 69090 includes a reference electrode, source 690104 ispreferably a potentiostat.

Controlled energy source 690106 is preferably a conventionalcontrollable electronic circuit driving device for controlling theoperation of sonication device 690105. Operation of source 690106 iscontrolled by microprocessor 690107. Microprocessor 690107 is aconventional processor device, such as a software-programmedmicroprocessor, a microcontroller, or the like. Microprocessor 690107controls the operation of detector 690102 and energy sources 690104 and690106, and receives intensity data from detector 690102 along withvoltage and/or current data from source 690104. Preferably,microprocessor 690107 is additionally capable of processing the assaydata and providing a corresponding output to a user and/or to anotherdevice.

In operation, a sample comprising reagents 69098 is introduced viasample inlet port 69095 into reaction enclosure 69094. The reagentsrequired for conducting an ECL assay may already have been added to thesample. Said reagents include: ECL coreagents (e.g., tripropylamine),ECL moieties (e.g., Ru(II)(bpy) 3 or derivatives, preferably linked toan analyte or the binding partner of an analyte), blocking agents (e.g.,BSA), buffers, exipients, additives and preservatives. In a preferredembodiment, the cartridge is prestored with some or all of the reagentsrequired to conduct an assay, shown as reagents 69097B. In an especiallypreferred embodiment, reagents 69097B are stored in a dry form withinreaction enclosure 69094.

To conduct an assay, cartridge 69090 is placed in instrument 690101,sonication device 690105 is structurally coupled to diaphragm 69092, anddevice 690105 activated by source 690106 to sonicate diaphragm 69092.Sonication energy is then transmitted through diaphragm 69092 toreagents 69098. Depending upon the mounting of diaphragm 69092,sonication energy may also be transmitted to base 69091 which willconduct such energy to reaction enclosure 69094, and thus to reagents69098.

The sonication causes reagents 69098 and reagents 69097B to mix,speeding the rate of reaction among components reagents 69098 and/or69097B and the rate of mass transfer of reagents 69098 and/or 69097B toand from diaphragm 69092. Sonication energy from device 690105significantly increases the rate of mass transfer of reagents 69098and/or 69097B to support 69092, thereby increasing the rate of bindingreactions between reagents 69097A and components of reagents 69097B and69098, and decreasing the time required to make an ECL measurement.Electrical energy is applied to diaphragm 69092 and to electrodes 69093,by source 690104 via connector 690103 and leads 690106, to cause anelectrochemiluminescent moiety in reactants 69097A, 69097B and/or 69098to luminesce. The light produced by the ECL reaction may be measured (orimaged) while sonication device 690105 operates or thereafter.

Microprocessor 690107 controls the operation of sources 690104 and690106 and receives intensity data from detector 690102 along withvoltage and/or current data from source 690104. Microprocessor 690107analyzes, and may store, the received data and preferably produces acorresponding output for provision to a user or to another device (notshown). Preferably, upon completion of data collection, microprocessor690107 notifies the user that cartridge 69090 may be removed frominstrument 690101. Upon receiving such notification from microprocessor690107, or otherwise determining that assay data collection is complete,the cartridge 69090 is removed from device 690101 and suitably disposedof or recycled.

In an alternate embodiment of system 690100, that portion of leads 69096coupled to diaphragm 69092 is omitted and an electrical connection isadded between source 690104 and sonication device 690105. Accordingly,the corresponding connection of connector 690103 may also be omitted. Inthis embodiment, sonication device 690105 functions as the electricalcorrection to diaphragm 69092. When cartridge 69090 is inserted intoinstrument 690101, electrical energy is provided through sonicationdevice 690105 to reagents 69098 via diaphragm 69092. Such application ofelectrical energy may or may not be simultaneous with the application ofsonication energy.

In an alternate embodiment, diaphragm 69092 and/or enclosure 69094 arepre-coated with a reagent or the like. Sonication of electrode 69092 maycause such reagent to loosen, allowing the reagent to mix with reagents69098 within enclosure 69094.

In another alternative embodiment, a dry reagent 69097B is prestored inreaction enclosure 69094 and liquid reagents 69098 are introduced intoreaction enclosure 69094 to directly contact dry reagent 69097B. Uponactivation of sonication device 690105, dry reagent 69097B and liquidreagent 69098 intermix at a significantly faster rate than in theabsence of sonication energy. The intermixed reagents may react e.g.,with each other and/or with reagents on a solid-phase support 69092, oranother reagent may then be added and also intermixed. In a differentembodiment, reagent 69097B is omitted.

The interior surfaces of reaction enclosure 69094 may become coated witha substance that interferes with an assay. This interfering substancemay include a contaminant, cellular debris, a non-specifically boundreagent, a reaction byproduct, or the like. In yet another embodiment ofthe invention, sonication device 690105 is activated and the sonicationenergy removes the interfering substances from the interior surfaces ofenclosure 69094 by sonicating such substances to loosen or by causingincreasing the rate of mass transport at the surfaces. For example, anECL assay may use cleaning cycles involving activation of device 690105before and/or after the binding reaction to properly prepare theelectrode for the excitation of ECL. These cleaning cycles may involveadding to reaction enclosure 69094 a cleaning solution which assists inloosening such interfering substances.

In still another alternate embodiment, sonication device 690105 andsource 690106 are omitted from instrument 690101 and diaphragm 69092additionally comprises a sonication device like device 690105. Further,source 690104 incorporates the functionality of source 690106.Electrical power from source 690104 to activate the sonication device ofdiaphragm 69092 is conducted via connector 690103 and leads 69096.

In continuous or intermittent ECL measurements, the rate of a bindingreaction is measured continuously or at intermittent intervals. Adescription of this process is found in U.S. Pat. No. 5,527,710(Nacamulli et al.). The present invention will act to increase the rateof binding reactions in such assays, and will also provide reproduciblemixing so as to provide precise and reproducible rate measurements.Sonication may also be continuous or intermittent during such assays. Anadvantage of continuous or intermittent measurements for determining therate of a binding reaction is that it offers increased sensitivity andprecision as compared to single-point ECL measurements.

6. EXAMPLES

All carbon fibrils used in these examples were CC fibrils obtained fromHyperion Catalysis Incorporated, Lot Number 166-39-1. These fibrils haddiameters that ranged approximately from 3.5 nm to 70 nm, with lengthsgreater than 10 times the diameter, an outer region of multiple layersof carbon atoms and a distinct inner core region.

6.1. Preparation of An MAB PMAMS Surface By Micro-Stamping

An exposed and developed photoresist master of 1-2 microns thickness isprepared according to well known procedures in a square array pattern. A10:1 mixture of SYLGARD silicone elastomer 184 (poly(dimethylsiloxane);available from Dow Corning) and the corresponding SYLGARD 184 curingagent is poured over the master and cured. The polymerized SYLGARD 184is carefully removed from the silicon master. The resulting elastomericstamp is “inked” by exposure to a hydrophilic OH-terminated terminatedalkane thiol, SH(CH₂)₁₁—(OCH₂CH₂)₆OH, in an ethanolic solution (1-10mM), robotically brought into pin registered contact with an alignedgold surface, and removed. The substrate is then washed for a fewseconds (e.g., 2-10 seconds) with a solution of a hydrophobicCH₃-terminated alkane thiol, SH(CH₂)₁₀CH₃ (1-10 mM in ethanol) (Kumar etal., supra and Prime et al., Science 252:1164-7). The resulting surfaceis then gently dried under a stream of nitrogen. A capillary arraycontaining hydrophilic solutions is then robotically brought into pinregistered contact with the aligned surface aligning the capillarieswith the SH(CH₂)₁₁—(OCH₂CH₂)₆OH domains. Each capillary in the capillaryarray contains monoclonal antibodies (MABs), specific for an analyte ofinterest, capable of covalently binding to the reactive OH groups on thehydrophilic domains through an amide linkage.

6.2. Preparation Of An MAB And Nucleic Acid PMAMS Surface ByMicro-Stamping

An exposed and developed photoresist master of 1-2 microns thickness isprepared according to well known procedures in a square array pattern. A10:1 mixture of SYLGARD silicone elastomer 184 and the correspondingSYLGARD 184 curing agent is poured over the master and cured. Thepolymerized SYLGARD 184 is carefully removed from the silicon master.The resulting elastomeric stamp is “inked” by exposure to a hydrophilicOH-terminated alkane thiol, SH(CH₂)₁₁—(OCH₂CH₂)₆OH, in an ethanolicsolution (1-10 mM), robotically brought into pin registered contact withan aligned gold surface, and removed. The substrate is then washed for afew seconds (e.g., 2-10 seconds) with a solution of a hydrophobicCH₃-terminated alkane thiol, SH(CH₂)₁₀CH₃ (1-10 mM in ethanol) (Kumar etal., supra and Prime et al., Science 252:1164-7). The resulting surfaceis then gently dried under a stream of nitrogen. A capillary arraycontaining hydrophilic solutions is then robotically brought into pinregistered contact with the aligned surface aligning the capillarieswith the SH(CH₂)₁₁—(OCH₂CH₂)₆OH domains. Each capillary in the capillaryarray contains antibodies or modified nucleic acids, specific for ananalyte of interest, capable of covalently binding to the reactive OHgroups on the hydrophilic domains through amide bond linkages.

6.3. Preparation Of A PMAMS Surface By Etching

A clean gold surface is exposed to a hydrophilic OH-terminated alkanethiol, SH(CH₂)₁₁—(OCH₂CH₂)₆OH (Prime et al., Science 252:1164-1167) inan ethanolic solution (1-10 mM). A linear array of fine tipped etchingutensils is robotically brought into optically registered contact withan aligned gold surface, and the linear array is used to etch in boththe X and Y dimensions of the surface creating a two dimensional gridarray of SH(CH₂)₁₁—(OCH₂CH₂)₆OH domains. The substrate is then washedfor a few seconds (eg. 2-10 seconds) with a solution of a hydrophobicSH(CH₂)₁₁—(OCH₂CH₂)₆CH₃ (1-10 mM in ethanol). The resulting surface isthen gently dried under a stream of nitrogen. A capillary arraycontaining hydrophilic solutions is then robotically brought into pinregistered contact with the surface aligning the capillaries with theSH(CH₂)₁₁—(OCH₂ CH₂)₆OH domains. Each capillary in the capillary arraycontains antibodies or nucleic acids, specific for an analyte ofinterest, capable of covalently binding to the reactive OH groups on thehydrophilic domains.

6.4. Sandwich Assay On A PMAMS Surface

A transparent PMAMS surface is made as described above which issubstantially transparent with a patterned multi-specific array ofprimary antibodies linked to the surface. The support, electrode assay,monologues surface use selected to be transparent. The PMAMS surface isthen exposed to a solution sample suspected of containing an analyte ofinterest to be assayed. The sample is then washed away leaving antibodybound analytes on the surface. The PMAMS surface is then exposed to asolution containing secondary ECL-labeled antibodies specific for boundanalytes on the surface. This solution is then washed from the PMAMSsurface leaving ECL labeled secondary antibodies bound to the domainswhere analyte is present.

The electrode assay is protected by a removable barrier to preventpremature contact of the sample with the electrode surface in order toavoid contamination effects. The barrier is then removed and theelectrode array, that is wetted with assay buffer, is brought intoaligned contact with the PMAMS surface. The electrode array is connectedto an electronic potential wave form generator, and potential is appliedto working electrode/counterelectrode pairs. A CCD then reads the lightemitted and the signal is sent to a microprocessor which converts thesignal to the desired readout form.

The readout is compared to the readout obtained using controls in theform of known quantities of an analyte of interest to calculate theactual quantity of analyte.

6.5. Assay On A First And Second PMAMS Surface

A transparent PMAMS surface is made as described above with a patternedmulti-specific array of primary antibodies linked to the surface. ThePMAMS surface is then exposed to a solution sample suspected ofcontaining an analyte of interest to be assayed. The sample is thenwashed away leaving antibody bound analytes on the surface.

A second PMAMS, under a protective cover, is provided, with analternating hydrophobic/hydrophilic pattern on which there are patternedmicro-drops of a plurality of secondary antibodies labeled with ECL tag.

The barrier protecting the second PMAMS in register with the first PMAMSis removed and the micro-drops are brought into register with theprimary antibody binding domains on the first PMAMS. The second PMAMS islifted off and the electrode array and is brought into aligned contactwith the first PMAMS surface. The electrode array is connected to anelectrical potential wave form generator, and potential is applied toworking electrode/counterelectrode pairs. A photo multiplier tube thenreads the light emitted and the signal is sent to a microprocessor whichconverts the signal to the desired readout form.

The readout is compared to the readout obtained using controls in theform of known quantities of an analyte of interest to calculate theactual quantity of analyte.

6.6. Nucleic Acid Assay On A PMAMS Surface

A transparent PMAMS surface is made as described above with a patternedmulti-specific array of single-stranded nucleic acid probes linked tothe surface. The probes are complementary to the 5′ region of a nucleicacid analyte of interest. The PMAMS surface is then exposed to asolution sample suspected of containing a hybridizable nucleic acidanalyte of interest to be assayed, the sample having been previouslydenatured, i.e., treated to render the analyte of interest singlestranded. The sample is then washed away leaving hybridized analytes onthe surface. The PMAMS surface is then exposed to a solution containingsecondary ECL labeled nucleic acid probes specific for the 3′ terminusof the nucleic acid analytes bound on the surface. This solution is thenwashed from the PMAMS surface leaving ECL labeled nucleic acid probesbound to the domains where analyte is present.

The barrier protecting the second PMAMS in register with the first PMAMSis removed and the micro-drops are brought into register with theprimary antibody binding domains on the first PMAMS. The second PMAMS islifted off and the electrode array and is brought into aligned contactwith the first PMAMS surface.

The electrode array is connected to an electronic potential wave formgenerator, and potential is applied to workingelectrode/counterelectrode pairs. A CCD then reads the light emitted andthe signal is sent to a microprocessor which converts the signal to thedesired readout form.

The readout is compared to the readout obtained using controls in theform of known quantities of an analyte of interest to calculate theactual quantity of analyte.

6.7. Competitive Assay On A PMAMS Surface With A PhotomultiplierDetector

A transparent PMAMS surface is made as described above with a patternedmulti-specific array of primary antibodies, specific for an analyte ofinterest, linked to the surface. The PMAMS surface is then exposed to asolution sample to be assayed which is a mixture of a sample suspectedof containing the analyte of interest and a known amount of an ECLlabeled molecule competitive with the analyte of interest for binding tothe antibodies. The sample is then washed away leaving antibody boundanalytes and/or labelled competitive binders on the surface.

The electrode array is protected by a removable barrier to preventcontact of the sample with the electrode surface in order to avoidcontamination effects. The barrier is then removed and the electrodearray, that is wetted with assay buffer, is brought into aligned contactwith the PMAMS surface. The electrode array is connected to a electronicpotential wave form generator, and potential is applied to workingelectrode/counterelectrode pairs. A photomultiplier tube then reads thelight emitted and the signal is sent to a microprocessor which convertsthe signal to the desired readout form.

The readout is compared to the readout obtained using controls in theform of known quantities of an analyte of interest to calculate theactual quantity of analyte.

6.8. Competitive Assay On A PMAMS Surface With A CCD Detector

A transparent PMAMS surface is made as described above with a patternedmulti-specific array of primary antibodies linked to the surface. ThePMAMS surface is then exposed to a solution sample suspected ofcontaining the analyte of interest to be assayed. The sample is thenwashed away leaving antibody bound analytes on the surface.

A second PMAMS, under a protective cover, is provided with analternating hydrophobic/hydrophilic pattern on which there are patternedmicro-drops of a plurality of a known amount of an ECL labeled moleculecompetitive with an analyte of interest.

The barrier protecting the second PMAMS in register with the first PMAMSis removed and the micro-drops are brought into register with theprimary antibody binding domains on the first PMAMS. The second PMAMS islifted off and the electrode array is brought into aligned contact withthe PMAMS surface. The electrode array is connected to a electronicpotential wave form generator, and potential is applied to workingelectrode/counterelectrode pairs. A CCD then reads the light emitted andthe signal is sent to a microprocessor which converts the signal to thedesired readout form.

The readout is compared to the readout obtained using controls in theform of known quantities of an analyte of interest to calculate theactual quantity of analyte.

6.9. Preparation Of An MAB PMAMS Surface By Micro Stamping With AnSH(CH₂)₁₀CH₃ Alkane Thiol

An exposed and developed photoresist master of 1-2 microns thickness isprepared according to well known procedures in a square array pattern. A10:1 mixture of SYLGARD silicone elastomer 184 (poly(dimethylsiloxane);available from Dow Corning) and the corresponding SYLGARD 184 curingagent is poured over the master and cured. The polymerized SYLGARD 184is carefully removed from the silicon master. The resulting elastomericstamp is “inked” by exposure to a hydrophilic OH-terminated alkanethiol, SH(CH2)₁₁OH, in an ethanolic solution (1-10 mM), roboticallybrought into pin registered contact with an aligned gold surface, andremoved. The substrate is then washed for a few seconds (e.g., 2-10seconds) with a solution of a hydrophobic CH₃-terminated alkane thiol,SH(CH₂)₁₀CH₃ (1-10 mM in ethanol) (Kumar et al., supra). The resultingsurface is then gently dried under a stream of nitrogen. A capillaryarray containing hydrophilic solutions is then robotically brought intopin registered contact with the aligned surface aligning the capillarieswith the SH(CH₂)₁₁OH domains to place specific antibodies at eachdomain. Each capillary in the capillary array contains monoclonalantibodies, specific for an analyte of interest, capable of covalentlybinding to the reactive OH groups on the hydrophilic domains.

6.10. Preparation Of An MAB And Nucleic Acid PMAMS Surface ByMicro-Stamping With An SH(CH₂)₁₀CH₃ Alkane Thiol

An exposed and developed photoresist master of 1-2 microns thickness isprepared according to well known procedures in a square array pattern. A10:1 mixture of SYLGARD silicone elastomer 184 and the correspondingSYLGARD 184 curing agent is poured over the master and cured. Thepolymerized SYLGARD 184 is carefully removed from the silicon master.The resulting elastomeric stamp is “inked” by exposure to a hydrophilicOH-terminated alkane thiol, SH(CH₂)₁₁OH, in an ethanolic solution (1-10mM), robotically brought into pin registered contact with an alignedgold surface, and removed. The substrate is then washed for a fewseconds (e.g., 2-10 seconds) with a solution of a hydrophobicCH₃-terminated alkane thiol, SH(CH₂)₁₀CH₃ (1-10 Mm in ethanol) (Kumar etal., supra). The resulting surface is then gently dried under a streamof nitrogen. A capillary array containing hydrophilic solutions is thenrobotically brought into pin registered contact with the aligned surfacealigning the capillaries with the SH(CH₂)₁₁OH, domains to place specificantibodies and/or hybridizable nucleic acids at each domain. Eachcapillary in the capillary array contains antibodies or modified nucleicacids, specific for an analyte of interest, capable of covalentlybinding to the reactive OH groups on the hydrophilic domains throughamide bond linkages.

6.11. Preparation Of A PMAMS Surface Using A Streptavidin-Biotin Linker

An exposed and developed photoresist master of 1-2 mm thickness isprepared according to well known procedures in a square array pattern. A10:1 mixture of SYLGARD silicone elastomer 184 and the correspondingSYLGARD 184 curing agent is poured over the master and cured. Thepolymerized SYLGARD 184 is carefully removed from the silicon master.The resulting elastomeric stamp is “inked” by exposure to a mixture ofmercaptoundecanol and12-mercapto(8-biotinamide-3,6-dioxaoctyl)dodecanamide where the molefraction of the biotinylated thiol is 0.1 (see Spinke et al., 1993,Langnuir 9:1821-5 and Spinke et al., 1993, J. Chem. Phys. 99(9):7012-9). The substrate is then washed for a few seconds (e.g., 2-10seconds) with a solution of a hydrophobic CH₃-terminated alkane thiol,HS(CH₂)₁₀CH₃ alkane thiol (1-10 mM in ethanol) (see Kumar et al. supra,Biebuyck, Whitesides). The resulting surface is then gently dried undera stream of nitrogen. A capillary array containing a solution ofstreptavidin in each capillary is then robotically brought into pinregistered contact with the aligned surface. Each capillary in thecapillary array is aligned and brought into contact with a biotinylateddomain and the capillary array is removed and the surface washed. Asecond capillary array containing a multiplicity of biotinylatedantibodies and biotinylated nucleic acids solutions is then roboticallybrought into pin registered contact with the aligned surface to placespecific antibodies and nucleic acids on each domain.

6.12. Preparation Of An MAB Single Surface

An electrode array of interdigitating working and counterelectrode pairson a gold on silicon surface is fabricated through methods known in theart (for example see Kumar et al supra). In this example, the electrodearray and the discrete binding domain array exist on the same surface ofa support. An exposed and developed photoresist master of 1-2 micronsthickness is prepared according to well known procedures in the patternof the working electrodes. A 10:1 mixture of SYLGARD silicone elastomer184 (poly(dimethylsiloxane(PDMS)); available from Dow Corning) and thecorresponding SYLGARD 184 curing agent is poured over the master andcured. The polymerized SYLGARD 184 is carefully removed from the siliconmaster. The resulting elastomeric stamp is “inked” by exposure to ahydrophilic OH-terminated alkane thiol, SH(CH₂)₁₁—(OCH₂CH₂)₆OH, in anethanolic solution (1-10 mM), robotically brought into pin registeredcontact with the aligned working electrodes on gold electrode arraysurface, and is then removed. A capillary array containing hydrophilicsolutions is then robotically brought into pin registered contact byaligning the capillaries with the SH(CH₂)₁₁—(OCH₂CH₂)₆OH domains on theelectrode array surface to place specific antibodies on each domain.Each capillary in the capillary array contains monoclonal antibodies,specific for an analyte of interest, capable of covalently binding tothe reactive OH groups on the hydrophilic domains through an amidelinkage.

6.13. Assay Conducted On An MAB Single Surface

A support as described 6.12, supra, is fabricated. A PDMS stamp isfabricated as previously described from a photoresist master patternedas rings which each independently circumscribe a workingelectrode/counterelectrode pair. The electrode array surface is thenexposed to a sample to be analyzed, washed with a mixture of ECLlabelled secondary antibodies, and then washed with an assay buffersolution containing tripropyl amine. The PDMS stamp is then aligned andbrought into registered contact aligning the rings of the PDMS stamp soas to circumscribe and define individual volume elements of assay bufferabove each electrode pair. An overpotential is applied to the electrodepairs so as to release the monolayer from the surface exposing theworking electrode to the ECL labelled secondary antibodies. Aphotomultiplier tube then reads the light emitted through thetransparent PDMS and the signal is sent to a microprocessor whichconverts the signal to the desired readout form.

The readout is compared to the readout obtained using controls in theform of known quantities of an analyte of interest to calculate theactual quantity of analyte.

6.14. Preparation Of A Single Surface With Working And Counterelectrodes

An electrode array of interdigitating working and counter gold electrodepairs with gold binding domains in between the interdigitatingelectrodes on a gold on silicon support is fabricated through methodsknown in the art (for example see Kumar et al. supra). In this example,the electrode array and the discrete binding domain array exist on thesame surface. An exposed and developed photoresist master of 1-2 micronsthickness is prepared according to well known procedures in the patternof the binding domains in between the interdigitating electrode pairs. A10:1 mixture of SYLGARD silicone elastomer 184(poly(dimethylsiloxane(PDMS)); available from Dow Corning) and thecorresponding SYLGARD 184 curing agent is poured over the master andcured. The polymerized SYLGARD 184 is carefully removed from the siliconmaster. The resulting elastomeric stamp is “inked” by exposure to ahydrophilic OH-terminated alkane thiol, SH(CH₂)₁₁—(OCH₂CH₂)₆OH, in anethanolic solution (1-10 mM), robotically brought into pin registeredcontact with the aligned gold binding domains on the electrode arraysurface, and is then removed. A capillary array containing hydrophilicsolutions is then robotically brought into pin registered contact,aligning the capillaries with the SH(CH₂)₁₁—(OCH₂CH₂)₆OH domains on theelectrode array surface to place specific antibodies on each domain.Each capillary in the capillary array contains antibodies specific foran analyte of interest, capable of covalently binding to the reactive OHgroups on the hydrophilic domains through an amide linkage.

6.15. Assay Conducted On A Single Surface With Working AndCounterelectrodes

A support surface as described 6.14 supra is fabricated by the describedmethods. The prepared surface is exposed to a sample to be analyzed,washed with a mixture of ECL labelled secondary antibodies, and thenwashed with an assay buffer solution containing tripropyl amine. Theelectrode array is connected to a electronic potential wave formgenerator, and potential is applied to workingelectrode/counterelectrode pairs. A photomultiplier tube then reads thelight emitted and the signal is sent to a microprocessor which convertsthe signal to the desired readout form.

The readout is compared to the readout obtained using controls in theform of known quantities of an analyte of interest to calculate theactual quantity of analyte.

6.16. Preparation Of A Surface With Counterelectrodes

An exposed and developed photoresist master of 1-2 microns thickness isprepared according to well known procedures in a square array pattern. A10:1 mixture of SYLGARD silicone elastomer 184 and the correspondingSYLGARD 184 curing agent is poured over the master and cured. Thepolymerized SYLGARD 184 is carefully removed from the silicon master.The resulting elastomeric stamp is “inked” by exposure to a hydrophilicOH-terminated alkane thiol, SH(CH₂)₁₁—(OCH₂CH₂)₆OH, in an ethanolicsolution (1-10 mM), robotically brought into pin registered contact withan aligned patterned counterelectrode and square binding domain on agold surface, and removed. The patterned gold surface consists ofaddressable ring counterelectrodes circumscribing the binding domainswhere the SH(CH₂)₁₁—(OCH₂CH₂)₆OH has been stamped. A gap or separationspace exists between each gold counterelectrode and each square goldsubstrate for each monolayer binding domain. A capillary arraycontaining binding reagent solutions is then robotically brought intopin registered contact with the aligned surface registering thecapillaries with the SH(CH₂)₁₁—(OCH₂CH₂)₆OH domains to place specificantibodies or nucleic acids on each domain. Each capillary in thecapillary array contains antibodies or nucleic acids, specific for ananalyte of interest, capable of covalently binding to the reactive OHgroups on the hydrophilic domains.

6.17. Assay Conducted On A Single Surface With The Working AndCounterelectrodes On Different Surfaces

The support surface described above in example 6.16 is exposed to asample solution to be analyzed. The support surface is then washed andexposed to a solution containing a plurality of ECL labelled monoclonalantibodies or ECL labelled nucleic acids of differing specificity andthen washed with assay buffer containing tripropyl amine. A transparentaddressable working electrode array is fabricated with each workingelectrode in the array corresponding to a discrete bindingdomain/counterelectrode region on the support as described above inSection 6.16. The two supports are wetted with the assay buffer androbotically brought into registered aligned conformal contact. Theelectrode arrays are connected to an electronic potential wave formgenerator, and potential is applied to the aligned workingelectrode/counterelectrode pairs creating a potential field between thetwo supports. A CCD then reads the light emitted through the transparentworking electrode and the signal is sent to a microprocessor whichconverts the signal to the desired readout form.

The readout is compared to the readout obtained using controls in theform of known quantities of an analyte of interest to calculate theactual quantity of analyte.

6.18. Fabrication Of A CC (Dispersed) Fibril MAT By Vacuum Filtration

An aqueous slurry of CC fibrils, with 1 mg fibrils/mL solution wasprepared by mixing 0.1% w/w CC fibrils/deionized water. The CC fibrilswere dispersed (the larger, micron-scale aggregates were dispersed intosmall aggregates or individual fibers) in the slurry by immersing a 400watt sonication horn in the slurry for between 10 minutes and 1 hour.The extent of dispersion was monitored by optical microscopy.

A nylon filter membrane (0.47 μm pore size, 25 mm diameter) was placedin a 25 mm diameter glass fritted filter. The dispersed fibril slurrywas filtered through the membrane/filter set-up by suction filtration(FIG. 23A). Aliquots of the slurry (5 ml) were diluted with 20 mldeionized water, then filtered through the membrane/filter set-up. Foran average mat of approximately 0.25-0.3 gram/cc, a mat of approximately100 μm required 6 aliquots.

Suction filtration was continued until all of the water from thedispersion was removed from the mat (by visual inspection). The mat waspeeled (by hand) directly from the filter membrane.

The mat was dried in an oven for approximately 10-15 minutes at 60° C.The mat was cut, punched or otherwise sectioned for use.

6.19. Fabrication of A Fibril Mat On A Metal Mesh Support By Evaporation

An aqueous slurry of CC fibrils, with 1 mg fibrils/mL solution wasprepared by mixing 0.1% w/w CC fibrils/deionized water. The CC fibrilswere dispersed (the larger, micron-scale aggregates were dispersed intosmall aggregates or individual fibers) in the slurry by immersing a 400watt sonication horn in the slurry for between 10 minutes and 1 hour.The extent of dispersion was monitored by optical microscopy.

A 1 cm² section of stainless steel mesh (400 count) was placed on a 25mm diameter paper filter. A 5 ml aliquot of the slurry was pipetted ontothe surface of the screen/filter paper ensemble. The water in the slurrywas allowed to evaporate, either at room temperature and pressure, or ina heated oven.

Once the fibril mat was dry, additional aliquots were added. The fibriland screen were peeled as a single unit from the filter paper.

The mat was cut, punched or otherwise sectioned for use.

6.20. Immobilization Of Avidin On Fibrils Bearing NHS-Ester FunctionalGroups

Fibrils derivatized with COOH (provided by Hyperion Catalysts Inc.) weresuspended in anhydrous dioxane at ˜10 mg/ml with constant stirring. A20-fold molar excess of N-hydroxysuccinimide was added and allowed todissolve. Next, a 20-fold molar excess ofethyl-diamino-propyl-carbodiimide (EDAC) was added, and the mixture wasstirred for 2 hours at room temperature.

After stirring, the supernatant was aspirated and the solids were washedthree times with anhydrous dioxane, one time with anhydrous methanol,and filtered on a 0.45 μm polysulfone membrane. The filtrate was washedwith additional methanol and the placed in a glass vial under vacuumuntil no further reduction in weight was observed.

10.4 mg of NHS-ester fibrils were washed with PBS-1 (˜70 mM phosphate,150 mM NaCl) (ORIGEN reagent 402-130-01, pH 7.8, IGEN, Inc.). The washedfibrils were suspended in 2.3 ml solution of avidin (8.3 mg avidin/mlPBS-1).

The suspension was allowed to sit at room temperature for 1.5 hours,with constant rotation of the flask to provide agitation.

After 1.5 hours, the suspension was stored for 16 hours at 4° C., thenbrought to room temperature and washed with PBS-1 and stored at 4° C. asa suspension in PBS-1.

6.21. Immobilization Of Monoclonal Antibody (Anti-AFP) On Carbon Fibrils

Carbon fibrils functionalized with NHS esters were prepared as describedin Example 6.20.

14 mg of fibril-NHS ester was mixed with 500 ml PBS-1 buffer. Themixture was sonicated for 20 min, until it became a viscous slurry. Anadditional 500 ml of PBS-1 buffer was added.

A total of 1.6 mg anti-AFP (alpha-fetal protein) antibody in 80 ml PBS-1was added to the above slurry. The reaction was allowed to sit at roomtemperature for 2.5 hours.

6 ml PBS-1 buffer was added and the reaction mixture was centrifuged at4° C. for 5 minutes. The supernatant was removed by pipette. Thisprocedure was repeated 9 times.

After the final wash, the supernatant was removed, and thefibril-antiAFP product was stored at 4° C.

6.22. Cyclic Voltammograms of Fibril Mats: Comparison Of Fibril Mat WithGold Foil Electrode

Cyclic voltammograms of 6 mM Fe^(3+/2+) (CN)₆ in 0.5 M K₂SO₄ weremeasured. In FIG. 30A, the CV for a plain fibril mat of CC(dispersed)was measured at 0.10 mA/cm at 10, 25 and 50 mV/sec. The mat wasfabricated as described in Example 6.18. In FIG. 30B, the CV wasmeasured for a gold foil electrode at 0.05 mA/cm at 10, 25 and 50mV/sec. All potentials are in Volts vs. Ag/AgCl.

6.23. Electrochemical Properties Of Fibril Mat Electrodes: Comparison OfAnodic Peak Current With Thickness Of The Mat

Cyclic voltammograms of 6 mM Fe^(3+/2+) (CN)₆ in 0.5 M K₂SO₄ weremeasured for fibril mats of the same geometric area (0.20 cm²), butdifferent thicknesses. The anodic peak current (FIG. 31) increased withincreasing thickness of mat for thicknesses that ranged from 24 μm to425 μm. For each thickness, the anodic peak current also increased withincreasing scan rates (for rates that ranged from 10 mV/sec to 150mV/sec). The rate of increase of the anodic peak current, as a functionof thickness, also increased with increasing thickness. Fibril mats thatwere 24 μm thick behaved comparably to gold foil electrodes.

6.24. Non-Specific Binding Of Proteins To Fibrils

Non-specific binding of proteins to carbon fibrils (cc) was measured asfollows: i) a solution of Ru(bipy)₃ ²⁺ (“TAG1”) labeled proteins wasexposed to a known quantity of carbon fibrils until equilibrium wasreached; ii) the labeled-protein/fibril solution was centrifuged, andthe supernatant was collected, and iii) the amount of labeled-proteinremaining in the supernatant was assayed using electrochemiluminescence(ECL).

To generate the curve shown in FIG. 32, anti-CEA antibody attached toderivatized TAG1 (antibody to carcinoembryonic antigen attached to aderivatized TAG1 ECL label) at 3 μg/mL, was added to serial dilutions ofCC(plain) fibrils in 0.1 M potassium phosphate buffer at pH 7. Fibrilswere removed by centrifugation after vortexing for 20 minutes. ECLassays that measured the amount of protein (unbound) remaining in thesupernatant were run in an ORIGEN 1.5 (IGEN, Inc.) analyzer on aliquotsof the reaction mixture supernatant diluted 5 times with ORIGEN assaybuffer. A decrease in the ECL lative to the ECL signal for an object ofthe reaction at had not been exposed to fibrils) resulted from bindingof protein labelled with a derivatized TAG1 her concentration of carbonfibrils were present.

6.25. Reduction Of Non-Specific Binding Of Proteins To Fibrils WithDetergents/Surfactants

Using the method described in Example 6.2.4, the effect of surfactant onprotein binding to fibrils was analyzed. Triton X-100 added to theanti-CEA attached to derivatized TAG1/fibril mixture, the solution wasincubated for 20 minutes, the tubes were centrifuged, and aliquots ofthe supernatant, diluted 5 times with ORIGEN assay buffer, were analyzedby ECL. The results are shown in the Table below and in FIG. 33. Tube[T-X100], Peak Prot-TAG1 [GF], Number ppm Intensity μg/ml ppm 19 16741611 2.65 52 18 837 1634 2.65 52 17 418 1697 2.65 52 16 209 1583 2.65 5215 105 1772 2.65 52 14 52 1463 2.65 52 13 26 627 2.65 52 12 13 23 2.6552A curve that results from plotting the ECL intensity of a proteinlabelled with a derivatized TAG1 in solution vs. Triton X-100concentration is shown in FIG. 33. A higher ECL signal corresponds tomore derivatized-TAG1-labeled protein in the supernatant, whichcorresponds to less derivatized-TAG1-labeled protein bound to thefibrils. Concentrations of Triton x-100 that ranged from 10 ppm to 100ppm reduced the extent of binding; increasing the concentration from 100to 2000 ppm did not further reduce the extent of binding.

6.26. ECL Of Free TAG In Solution With Fibril Mat Electrode

A fibril mat prepared as in Example 6.18 was installed in the mountingarea 3403 of the working electrode holder 3401 of the “Fibril Cell”fixture shown in FIG. 34. The holder 3401 was slipped into the bottom ofthe electrochemical cell compartment 3400. The 3 M Ag/AgCl referenceelectrode (Cyprus # EE008) was installed into the electrochemical cellcompartment through the reference cell hole 3402. The cell was filledwith Assay Buffer (IGEN # 402-005-01 lot# 5298) and attached to the PMTholder 3404. Using a EG&G PARC model 175 universal programmer and anEG&G model 175 Potentiostat/Galvanostat the potential was swept from 0 Vto +3 V vs. Ag/AgCl at 100 mV/s. The ECL was measured by a HamamatsuR5600U-01 which was powered at 900V by a Pacific Instruments model 126Photometer. The analog data was digitized at 10 Hz by a CIO-DAS-1601 A/Dboard driven by HEM Snap-Master. The Fibril Cell was drained, flushedwith 1000 pM TAG1 (IGEN # 402-004-C lot# 4297), and filled with 1000 pMTAG1. The potential was swept as with Assay Buffer. Shown in FIG. 35 arethe ECL traces (measured at 24.0±0.2 C) for Assay Buffer 3501 and 1000pM TAG1 3502. The dark corrected ECL peak area was 22.10 nAs for AssayBuffer and 46.40 nAs for 1000 pM TAG1.

6.27. ECL Of Adsorbed Labeled Antibody With Fibril Mat Electrode

Fibril mats were made to a thickness of 0.0035 inches from plaincc-dispersed fibrils in the manner described in Example 6.18. The driedmats were then punched into 3 mm disks and mounted onto supports. Thesupports used in this experiment were fabricated from 0.030 inchespolyester sheet patterned by screen printed conductive gold ink. Thisconductive gold ink formed the counter electrode, reference electrode,and provided leads for the working and other electrodes. Two fibril matdisks were mounted to each patterned support using two sided carboncontaining conductive tape (Adhesives Research). After mounting, thedisks were spotted with 0.5 μl of 10 μg/ml anti-TSH antibody attached toderivatized TAG1 in deionized water (Ru-TSH mono 1:2 26 JUN. 1995, IGEN,Inc.) or 0.5 μl of 10 μg/ml anti-TSH unTAG1′ ed capture antibody indeionized water (TSH poly 25 JUN . 1995, IGEN, Inc.) and allowed to dry.After drying, the mats were flooded with IGEN assay buffer. Flooded matson supports were loaded into an IGEN Origen 1.5 based instrument and ECLwas read using a scan rate of 500 mV/s from 0 to 4500 mV. FIG. 43compares the peak ECL signals from TAG1-antibody containing mats 4301and unTAG1′ ed capture antibody containing mats 4302.

6.28. ECL Using Fibril Mat Electrode For Sandwich Assay

Anti-AFP capture antibody was immobilized on fibrils as described above.Anti-AFP fibrils were washed into deionized water (dI) and resuspendedat a density of 1 mg/ml. A four layer fibril mat was produced usingvacuum filtration as described in Example 6.18. Two milligrams ofanti-AFP fibrils were added to 3 mg of plain CC dispersed fibrils andthe mixture diluted to a total volume of 20 ml in dI. The dilutedmixture was filtered onto a 0.45 μm nylon filter. This initial mat layerwas then followed by two core layers, each consisting of 5 mg of plainCC dispersed fibrils. The mat core was then topped with a mixed fibrillayer identical to the initial layer. This resulted in a fibril mat thatwas ˜40% anti-AFP fibrils on the top and bottom surface and ˜100% plainfibrils in the core. This mixed mat was air dried under vacuum andpunched into 3 mm disks. These disks were then mounted onto supports asdescribed in Example 6.27. Dry, supported, anti-AFP mats were floodedwith AFP calibrators A, C, and F (IGEN, Inc.) and allowed to incubatefor 15 minutes at room temperature on the bench top. After incubation,supported electrodes were washed with a dI stream for 10 seconds andthen blotted dry with a lint free wipe. Fibril mats were then floodedwith anti-AFP attached to antibody labelled with derivatized TAG1 (IGEN,Inc.) and allowed to incubate for 15 minutes at room temperature on thebench top. After incubation the supported electrodes were washed with dIand dried with a wipe. Fibril mats were then flooded with IGEN assaybuffer and read as described in Example 6.27.

6.29. ECL Detection Of TAG1-Labeled Avidin On A Polyacrylamide Surface

A cross-linked polyacrylamide gel containing covalently bound biotin wasprepared by copolymerization of acrylamide, bis-acrylamide, andN-Acryloyl-N′-biotinyl-3,6-dioxaoctane-1,9-diamine (biotin linked to anacrylamide moiety through a tri(ethylene glycol) linker) using wellknown conditions (initiation with ammonium persulfate and TEMED). Inthis experiment, the concentrations of the three monomeric species were2.6 M, 0.065 M, and 0.023 M respectively (these concentrations ofacrylamide and bis-acrylamide are reported to result in gels with poresizes smaller than most proteins). Polymerization of the solutioncontaining the monomers between two glass plates held apart to adistance of approximately 0.7 mm led to the formation of a slab gel withthe same thickness. After the polymerization reaction was complete, anyunincorporated biotin was washed out by soaking the gel in four changesof PBS. Avidin labeled with a derivatized TAG1 (where Avidin refers toNeutrAvidin, a modified avidin designed to exhibit reduced NSB, was usedin this experiment) was bound to the surface of the gel by soaking thegel in a solution containing the protein at a concentration of 50 μg/mLin PBS for 20 min. Excess TAG1-labeled avidin was then washed away bysoaking the gel in four changes of ECL assay buffer (200 mM sodiumphosphate, 100 mM tripropylamine, 0.02% (w/v) Tween-20, pH 7.2). Asshown in FIG. 39, the gel (3900) was then placed in contact with goldworking (3901) and counter (3902) electrodes patterned on a glasssupport (3903). Ramping the potential across the two electrodes from 0.0to 3.0 V and back to 0.0 V at a rate of 500 mV/s led to an ECL lightsignal as measured from a PMT (3904) placed above the gel (FIG. 40). Agel prepared without inclusion of the biotin containing acrylamidederivative gave no ECL signal (FIG. 41). This signal obtained for thebiotin-containing polymer was indicative of close to a full monolayer ofprotein is present on the surface of the gel.

6.30. ECL Sandwich Immunoassay On A Polyacrylamide Surface

A cross-linked polyacrylamide gel containing covalently bound biotin isprepared as described in Example 6.29. Streptavidin is adsorbed on thesurface of the gel to form a binding domain capable of capturing biotinlabeled species. The surface is treated with a solution containingtripropylamine, an unknown concentration of an analyte, a biotin-labeledantibody against the analyte, and a different ECL TAG1-labeled antibodyagainst the analyte. The presence of the analyte causes the formation ofa complex of the analyte and the two antibodies which is then capturedon the streptavidin surface. ECL tag bound to secondary antibody presenton the surface is measured as described in example 6.29

6.31. Multiple ECL Sandwich Immunoassays on Polyacrylamide SurfacesSupported On An Electrode

An exposed and developed photoresist master of 1-2 microns thickness isprepared according to well known procedures to give a pattern ofcircular depressions arranged in an array. A 10:1 mixture of SYLGARDsilicone elastomer 184 and the corresponding SYLGARD 184 curing agent ispoured over the master and cured. The polymerized SYLGARD is carefullyremoved from the silicon master. The resulting elastomeric stamp is“inked” by exposure to a solution containing the hydroxyl terminatedthiol HS—(CH₂)₁₁—(OCH₂CH₂)₃—OH (1-10 mM) in ethanol, brought intocontact with an aligned gold substrate and removed. The substrate iswashed for several seconds with a solution containing the thiolHS—(CH₂)₁₀—CH₃ (1-10 mM in ethanol). The resulting surface is thenrinsed with ethanol and dried under a stream of nitrogen. Treatment ofthe surface with a solution containing acryloyl chloride andtriethylamine in dioxane leads to functionalization of the hydroxylterminated domains with acrylate groups. A capillary array containingmixtures of acrylamide, bis-acrylamide, N-acryloylsuccinimide,azo-bis-cyanovaleric acid, and antibodies presenting amino groups isthen brought into contact with the aligned surface aligning thecapillaries with the acrylate terminated domains to place prepolymersolutions containing specific antibodies at each domain. Each capillaryin the capillary array contains antibodies specific for a differentanalyte of interest. Exposure of the patterned prepolymer droplets to UVlight leads to the formation of cross-linked gels on the substrate eachpresenting a binding domain at the surface. The assay is carried out bytreatment of the substrate with a mixture of analytes capable of bindingat one or more of the binding domains presented on the gel surfaces in abuffered solution containing tripropylamine and ECL-TAG1 labeledsecondary antibodies. The binding domains (4200, 4201, 4202) (onpolyacrylamide drops (4203) on a gold electrode (4232) are then placedin close proximity to an ITO working electrode (4204) as shown in FIGS.42A-B. Light emitted from each of the binding domains is quantifiedusing a CCD camera (4205) and compared to binding domains for internalstandards included in the sample solution.

6.32. Multiple ECL Competitive Immunoassays On Polyacrylamide SurfacesSupported On An Electrode

An exposed and developed photoresist master of 1-2 microns thickness isprepared according to well known procedures to give a pattern ofcircular depression arranged in an array. A 10:1 mixture of SYLGARDsilicone elastomer 184 and the corresponding SYLGARD 184 curing agent ispoured over the master and cured. The polymerized SYLGARD is carefullyremoved from the silicon master. The resulting elastomeric stamp is“inked” by exposure to a solution containing the hydroxyl terminatedthiol HS—(CH₂)₁₁—(OCH₂CH₂)₃—OH (1-10 mM) in ethanol, brought intocontact with an aligned gold substrate and removed. The substrate iswashed for several seconds with a solution containing the thiolHS—(CH₂)₁₀—CH₃ (1-10 mM in ethanol). The resulting surface is thenrinsed with ethanol and dried under a stream of nitrogen. Treatment ofthe surface with a solution containing acryloyl chloride andtriethylamine in dioxane leads to functionalization of the hydroxylterminated domains with acrylate groups. A capillary array containingmixtures of acrylamide, bis-acrylamide, N-acryloylsuccinimide,azo-bis-cyanovaleric acid, and antibodies is then brought into contactwith the aligned surface aligning the capillaries with the acrylateterminated domains to place prepolymer solutions containing specificantibodies at each domain. Capillaries in the capillary array containantibodies specific for different analytes of interest. Exposure of thepatterned prepolymer droplets to UV light leads to the formation ofcross-linked gels on the substrate each presenting a binding domain atthe surface. The assay is carried out by treatment of the substrate witha mixture of analytes capable of binding at one or more of the bindingdomains presented on the gel surfaces in a buffered solution containingtripropylamine and ECL-TAG1 labeled analogues of the analytes (i.e.,setting up a competition of ECL-TAG1 labeled and unlabeled analytes forbinding to the binding domains). The binding domains (4200, 4201 and4202) (on polyacrylamide drops (4203) on a gold electrode (4232)) arethen placed in close proximity to an ITO working electrode (4204) asshown in FIG. 42. Light emitted from each of the binding domains isquantified using a CCD camera (4205) and compared to binding domains forinternal standards included in the sample solution.

6.33. Multiple ECL Assays For Binding Of Cells On PolyacrylamideSurfaces Supported On An Electrode

An exposed and developed photoresist master of 1-2 microns thickness isprepared according to well known procedures to give a pattern ofcircular depressions arranged in an array. A 10:1 mixture of SYLGARDsilicone elastomer 184 and the corresponding SYLGARD 184 curing agent ispoured over the master and cured. The polymerized SYLGARD is carefullyremoved from the silicon master. The resulting elastomeric stamp is“inked” by exposure to a solution containing the hydroxyl terminatedthiol HS—(CH₂)₁₁—(OCH₂CH₂)₃—OH (1-10 mM) in ethanol, brought intocontact with an aligned gold substrate and removed. The substrate iswashed for several seconds with a solution containing the thiolHS—(CH₂)₁₀—CH₃ (1-10 mM in ethanol). The resulting surface is thenrinsed with ethanol and dried under a stream of nitrogen. Treatment ofthe surface with a solution containing acryloyl chloride andtriethylamine in dioxane leads to functionalization of the hydroxylterminated domains with acrylate groups. A capillary array containingmixtures of acrylamide, bis-acrylamide, N-acryloylsuccinimide,azo-bis-cyanovaleric acid, and antibodies directed against cell surfacesis then brought into contact with the aligned surface aligning thecapillaries with the acrylate terminated domains to place prepolymersolutions at each domain. Exposure of the patterned prepolymer dropletsto UV light leads to the formation of cross-linked gels on the substrateeach presenting a binding domain at the surface. The assay is carriedout by treatment of the binding domains first with a suspension ofcells, then with a mixture of binding reagents capable of binding one ormore of the cells bound to the gel surfaces in a buffered solutioncontaining tripropylamine and ECL-TAG1 labeled secondary antibodiesand/or other binding reagents specific for the analytes. The bindingdomains (4200, 4201, and 4202) (on polyacrylamide drops (4203) on a goldelectrode (4232) are then placed in close proximity to an ITO workingelectrode (4204) as shown in FIG. 42. Light emitted from each of thebinding domains is quantified using a CCD camera (4205) and compared tobinding domains for internal standards included in the sample solution.

6.34. Multiple ECL Assays For Binding Of Analytes To Cells OnPolyacrylamide Surfaces Supported On An Electrode

An exposed and developed photoresist master of 1-2 microns thickness isprepared according to well known procedures to give a pattern ofcircular depressions arranged in an array. A 10:1 mixture of SYLGARDsilicone elastomer 184 and the corresponding SYLGARD 184 curing agent ispoured over the master and cured. The polymerized SYLGARD is carefullyremoved from the silicon master. The resulting elastomeric stamp is“inked” by exposure to a solution containing the hydroxyl terminatedthiol HS—(CH₂)₁₁—(OCH₂CH₂)₃—OH (1-10 mM) in ethanol, brought intocontact with an aligned gold substrate and removed. The substrate iswashed for several seconds with a solution containing the thiolHS—(CH₂)₁₀—CH₃ (1-10 mM in ethanol). The resulting surface is thenrinsed with ethanol and dried under a stream of nitrogen. Treatment ofthe surface with a solution containing acryloyl chloride andtriethylamine in dioxane leads to functionalization of the hydroxylterminated domains with acrylate groups. A capillary array containingmixtures of acrylamide, bis-acrylamide, N-acryloylsuccinimide,azo-bis-cyanovaleric acid, and cells is then brought into contact withthe aligned surface aligning the capillaries with the acrylateterminated domains to place prepolymer solutions containing specificcell types at each domain. Capillaries in the capillary array containcells with different surface structures that bind different analytes.Exposure of the patterned prepolymer droplets to UV light leads to theformation of cross-linked gels on the substrate each presenting abinding domain at the surface. The assay is carried out by treatment ofthe gels with a sample containing a mixture of analytes capable ofbinding at one or more of the binding domains presented on the gelsurfaces in a buffered solution containing tripropylamine and ECL-TAGlabeled antibodies and/or other binding reagents specific for theanalytes. The binding domains (4200, 4201 and 4202) (on polyacrylamidedrops (4203) on a gold electrode (4232) are then placed in closeproximity to an ITO working electrode (4204) as shown in FIG. 42. Lightemitted from each of the binding domains is quantified using a CCDcamera (4205) and compared to binding domains for internal standardsincluded in the sample solution.

6.35. Multiple ECL Competitive Hybridization Assays On PolyacrylamideSurfaces Supported On An Electrode

An exposed and developed photoresist master of 1-2 microns thickness isprepared according to well known procedures to give a pattern ofcircular depression arranged in an array. A 10:1 mixture of SYLGARDsilicone elastomer 184 and the corresponding SYLGARD 184 curing agent ispoured over the master and cured. The polymerized SYLGARD is carefullyremoved from the silicon master. The resulting elastomeric stamp is“inked” by exposure to a solution containing the hydroxyl terminatedthiol HS—(CH₂)₁₁—(OCH₂CH₂)₃—OH (1-10 mM) in ethanol, brought intocontact with an aligned gold substrate and removed. The substrate iswashed for several seconds with a solution containing the thiolHS—(CH₂)₁₀—CH₃ (1-10 mM in ethanol). The resulting surface is thenrinsed with ethanol and dried under a stream of nitrogen. Treatment ofthe surface with a solution containing acryloyl chloride andtriethylamine in dioxane leads to functionalization of the hydroxylterminated domains with acrylate groups. A capillary array containingmixtures of acrylamide, bis-acrylamide, N-acryloylsuccinimide,azo-bis-cyanovaleric acid, and nucleic acid probes functionalized withamino groups is then brought into contact with the aligned surface,aligning the capillaries with the acrylate terminated domains to placeprepolymer solutions containing specific probes at each domain.Capillaries in the capillary array contain probes specific for a nucleicacid sequence of interest. Exposure of the patterned prepolymer dropletsto UV light leads to the formation of cross-linked gels on thesubstrate, each presenting a binding domain at the surface. The assay iscarried out by treatment of the substrate with a sample mixture that maycontain sequences capable of binding at one or more of the bindingdomains presented on the gel surfaces in a buffered solution containingtripropylamine and ECL-TAG1 labeled sequences which can compete with theanalytes of interest for binding to the surface. The binding domains(4200, 4201, and 4202) (on polyacrylamide drops (4203) on a goldelectrode (4232) are then placed in close proximity to an ITO workingelectrode (4204) as shown in FIG. 42. Light emitted from each of thebinding domains is quantified using a CCD camera (4205) and compared tobinding domains for internal standards included in the sample solution.

6.36. Multiple ECL Hybridization Sandwich Assays On PolyacrylamideSurfaces Supported On An Electrode

An exposed and developed photoresist master of 1-2 microns thickness isprepared according to well known procedures to give a pattern ofcircular depression arranged in an array. A 10:1 mixture of SYLGARDsilicone elastomer 184 and the corresponding SYLGARD 184 curing agent ispoured over the master and cured. The polymerized SYLGARD is carefullyremoved from the silicon master. The resulting elastomeric stamp is“inked” by exposure to a solution containing the hydroxyl-terminatedthiol HS—(CH₂)₁₁—(OCH₂CH₂)₃—OH (1-10 mM) in ethanol, brought intocontact with an aligned gold substrate and removed. The substrate iswashed for several seconds with a solution containing the thiolHS—(CH₂)₁₀—CH₃ (1-10 mM in ethanol). The resulting surface is thenrinsed with ethanol and dried under a stream of nitrogen. Treatment ofthe surface with a solution containing acryloyl chloride andtriethylamine in dioxane leads to functionalization of the hydroxylterminated domains with acrylate groups. A capillary array containingmixtures of acrylamide, bis-acrylamide, N-acryloylsuccinimide,azo-bis-cyanovaleric acid, and nucleic acid probes functionalized withamino groups is then brought into contact with the aligned surface,aligning the capillaries with the acrylate terminated domains to placeprepolymer solutions containing specific probes at each domain.Capillaries in the capillary array contain probes specific for a nucleicacid sequence of interest. Exposure of the patterned prepolymer dropletsto UV light leads to the formation of cross-linked gels on thesubstrate, each presenting a binding domain at the surface. The assay iscarried out by treatment of the substrate with a sample mixture that maycontain sequences capable of binding at one or more of the bindingdomains presented on the gel surfaces in a buffered solution containingtripropylamine and ECL-TAG1 labeled sequences which can bind theanalytes at sequences not complementary to the surface-bound probes. Thebinding domains (4200, 4201, and 4202) (on polyacrylamide drops (4203)on a gold electrode (4232) are then placed in close proximity to an ITOworking electrode (4204) as shown in FIG. 42. Light emitted from each ofthe binding domains is quantified using a CCD camera (4205) and comparedto binding domains for internal standards included in the samplesolution.

6.37. Multiple Assays Of Different Types on A Polyacrylamide SurfacesSupported On An Electrode

An exposed and developed photoresist master of 1-2 microns thickness isprepared according to well known procedures to give a pattern ofcircular depressions arranged in an array. A 10:1 mixture of SYLGARDsilicone elastomer 184 and the corresponding SYLGARD 184 curing agent ispoured over the master and cured. The polymerized SYLGARD is carefullyremoved from the silicon master. The resulting elastomeric stamp is“inked” by exposure to a solution containing the hydroxyl terminatedthiol HS—(CH₂)₁₁—(OCH₂CH₂)₃—OH (1-10 MM) in ethanol, brought intocontact with an aligned gold substrate and removed. The substrate iswashed for several seconds with a solution containing the thiolHS—(CH₂)₁₀—CH₃ (1-10 mM in ethanol). The resulting surface is thenrinsed with ethanol and dried under a stream of nitrogen. Treatment ofthe surface with a solution containing acryloyl chloride andtriethylamine in dioxane leads to functionalization of the hydroxylterminated domains with acrylate groups. A capillary array containingmixtures of acrylamide, bis-acrylamide, N-acryloylsuccinimide,azo-bis-cyanovaleric acid, and any of the binding reagents described inExamples 6.31-6.36 is then brought into contact with the aligned surfacealigning the capillaries with the acrylate terminated domains to placeprepolymer solutions containing specific probes at each domain. Eachcapillary in the capillary array contains binding domains specific foranalytes of interest. Exposure of the patterned prepolymer droplets toUV light leads to the formation of cross-linked gels on the substrateeach presenting a binding domain at the surface. The assay is carriedout by treatment of the substrate with a sample mixture that may containanalytes capable of binding at one or more of the binding domainspresented on the gel surfaces in a buffered solution containingtripropylamine and either ECL-TAG1 labeled analogues of analytes whichcompete with analytes for binding to the binding domains and/or ECL-TAG1labeled secondary binding reagents to the analytes of interest. Thebinding domains (4200, 4201, and 4202) (on polyacrylamide drops (4203)on a gold electrode (4232) are then placed in close proximity to an ITOworking electrode (4204) as shown in FIG. 42. Light emitted from each ofthe binding domains is quantified using a CCD camera (4205) and comparedto binding domains for internal standards included in the samplesolution.

6.38. Highly Reversible ECL

Polycrystalline gold electrodes (purchased from Bio-Analytical Services,2 mm²) were cleaned by hand polishing sequentially with 0.5 μm and 0.03μm alumina slurry, followed by chemical etching in 1:3 H₂O₂/H₂SO₄ andelectrochemical cycling in dilute H₂SO₄ between −0.2 V and 1.7 V vs.Ag/AgCl. The clean electrodes were then immersed overnight in a dilutesolution of octylthiol (C₈SH) dissolved in ethanol. Protein adsorptionwas carried out by covering C₈SH-modified electrodes with 20 μl ofTAG1-labeled bovine serum albumin (BSA) dissolved in phosphate buffersaline (PBS, 0.15 M NaCl/0.1 M NaPi, pH 7.2) and washing the surfaceextensively with the same buffer after ten minute incubation.

ECL was done in a three-electrode cell with a Ag/AgCl referenceelectrode, platinum wire counter electrode and an EG&G 283 potentiostat.The light intensity was measured with a Pacific Instruments photometerand a Hamamatsu photo-multiplier tube placing at the bottom of theelectrochemistry cell. The protein-adsorbed electrode was immersed in asolution of 0.1 M TPA and 0.2 M phosphate, pH 7.2. Highly reversible ECLresponse (substantially similar intensity on the forward and backwardscans) was observed when the electrode potential was cycled between 0.0V and 1.2 V, as shown in FIG. 44A, indicating the reversible nature ofthe ECL process and stability of the thiol and protein layers on theelectrode.

Cyclic voltammetric experiments were conducted on the same instrumentsas for ECL, without the use of PMT and photometer. In the experiment, aC₈SH-covered electrode (no protein) was placed in a solution of 1 mMpotassium ferricyanide (in PBS) and the electrode was scanned from +0.5V to 1.2 V and back, followed by another cycle between +0.5 V and −0.3V. It is indicative that the monolayer is still intact and not desorbingat 1.2 V, since there was only capacitive current in the voltammogrambetween +0.5 V and −0.3 V and no faradaic current of ferricyanide (FIG.44B).

6.39. Ouasi-Reversible ECL

Electrode modification and protein adsorption were done in the same wayas described above. In the ECL experiments, the potential was scannedbetween 0.0 V and 1.5 V, and the corresponding light intensity wasrecorded. As illustrated in FIG. 45A, there was some loss of ECL betweenthe forward and backward scans of the same cycle, as well as betweendifferent cycles. Cyclic voltammograms of the thiol/Au in ferricyanideafter oxidizing at 1.5 V showed a significant amount of faradaiccurrent, indicative of at least partial desorption of the thiolmonolayer at 1.5 V (FIG. 45B).

6.40. Irreversible ECL

In these experiments, electrode modification and protein adsorption wereconducted in the same way as in Example 6.38. To measure ECL, theelectrode potential was scanned all the way up to 2.0 V and back to 0.0V. Intense light was observed on the forward scan (more light than wasobserved under reversible conditions in Example 6.38), but it dropped tothe background on the reverse scan, as shown in FIG. 46A. Cyclicvoltammograms of the modified electrode in ferricyanide after oxidizingat 2.0 V indicated most of the thiol monolayer was desorbed (FIG. 46B).

6.41. An ECL Sandwich Immunoassay Using A Primary Antibody ImmobilizedOn A Patterned Gold Electrode

In this example an antibody against prostrate specific antigen (PSA) isimmobilized on a patterned gold electrode for use in an immunoassay forPSA.

An exposed and developed photoresist master of one to two micronsthickness is prepared according to well known procedures to give a layerof photoresist on a silicon support with a 1 mm×1 mm square patch wherephotoresist is removed. A 10:1 mixture of SYLGARD silicone elastomer 184and the corresponding curing agent is poured over the master and cured.The polymerized SYLGARD is carefully removed from the silicon master.The resulting elastomeric “stamp” is “inked” by exposing it to asolution containing the hydroxyl-terminated thiolHS—(CH₂)₁₁(OCH₂CH₂)₃—OH and the nitrilotriacetic acid (NTA)terminated-thiol HS—(CH₂)₁₁(OCH₂CH₂)₃OC(O)NH(CH₂)₄CH(CO₂H)N(CH₂CO₂H)₂ inethanol. The “inked” stamp is brought into contact with a gold substrateand removed to form a 1 mm×1 mm SAM. The substrate is washed for severalseconds with a solution containing only the hydroxl-terminated thiol inethanol, to prevent non-specific binding of proteins to the regionsoutside the stamped feature. The resulting surface is then rinsed withethanol and dried under a stream of nitrogen. Treatment of the surfacewith a solution of NiCl₂ followed by treatment with a solutioncontaining a fusion protein presenting the binding sites of an anti-PSAmouse monoclonal and the peptide (His)₆, leads to immobilization of thefusion protein on the surface in a controlled manner. This processyields a reproducible and predetermined amount of immobilized protein onthe surface. The orientation of the protein on the surface is controlledby the location of the (His)₆ sequence in the primary structure of thefusion protein. The absolute amount of immobilized protein is controlledby the ratio of NEA terminated-thiol to hydroxy-terminated thiol in thestamped SAM and by the surface area of the stamped feature. Acalibration curve for PSA is determined by preparing solutionscontaining known concentrations of PSA in serum (at concentrationsranging from 1 fM to 1 uM. A number of surfaces prepared as describedabove are treated with the PSA calibration standards and then with asolution containing a secondary antibody against PSA (labeled with aderivative of TAG1) at an optimized concentration. A calibration curveis determined by immersing the surfaces in a solution containing 0.1 MTPA and 0.2 M phosphate (pH 7.2), and measuring the peak intensity oflight emitted when the electrical potential at the gold surface iscycled between 0.0 and 2.0 V at a scan rate of 0.5V/sec. Thedetermination of unknown concentrations of PSA in serum in a sample isconducted by the same procedure except that the concentration of PSA iscalculated from the peak ECL signal by reference to the calibrationcurve.

6.42. Preparation of Aerosil-200 Silica Particles Coated WithStreptavidin

Aerosil-200 (Degussa Corporation, Akron, Ohio, USA), a fumed silica witha particle size of 12 nm and an active surface area of 175-220 m²/g, waschemically modified to introduce NHS ester groups. The modificationinvolved three steps: i) amino groups were introduced to the surface ofthe particles by reaction with 3-aminopropyltrimethoxysilane. TheAerosil-200 (155.5 mg) was combined with 3-aminopropyltrimethoxysilane(513 mg) in 5 mL of toluene and refluxed for 1 hour. The silicaparticles were washed two times with 4 mL of toluene, three times with 4mL of methanol and three times with 4 mL of dichloromethane. Each washconsisted of centrifugation of the suspension followed by resuspensionof the pellet in fresh solvent; ii) the amino groups were reacted withsuccinic anhydride to introduce carboxylic acid groups to the surface.The 55.2 mg of washed particles were resuspended in 3 mL of anhydrousDMF. Succinic anhydride (102 mg) and triethylamine (0.025 mL) were addedand the suspension stirred for 16 h. Additional succinic anhydride (50mg) was added and the reaction was allowed to proceed for another 3hours. Excess reagent was removed by washing the particles two timeswith DMF, once with methanol, and two times with dichloromethane; andiii) the carboxylic acid groups on the surface were activated asN-hydroxysuccinimide (NHS) esters by reaction with NHS andethyl-3-diaminopropylcarbodiimide (EDC). The particles (25.1 mg wereresuspended in 3 mL of methylene chloride. NHS (64 mg) and EDC (110 mg)were added and the suspension was then stirred for 3 hours. Theparticles were then washed three times with dichloromethane and driedunder vacuum.

The activated silica particles were coated with streptavidin by thereaction of the protein with the NHS esters on the surface of theactivated particles. The particles (˜1.5 mg) were added to solutioncontaining 0.750 mg of streptavidin in phosphate buffered saline (PBS),pH 7.85. The reaction was allowed to proceed for 16 hours. The particleswere then washed with PBS and water and resuspended in PBS to give astock suspension containing particles at a concentration of 0.1 mg/mL.

6.43. Formation Of A Fibril Mat on A Support Of Stainless Steel FilterPaper For Use In Particle-based ECL Assays

A fibril ink was formed by the sonication of a suspension of CC fibrils(0.1 mg/mL in 0.2% triton X-100 in water) for 15 minutes using a probesonicator (Branson Ultrasonic Corp., Danbury, Conn., USA). Thesuspension was filtered using gentle suction onto an ⅛″ diametercircular region on a disc of stainless steel filter paper (GA-4, BaekartFibre Technologies). The thickness of mats formed by this method wasapproximately 1 μm/mL of added fibril ink.

6.44. A Particle-based ECL Assay For AFP Using Streptavidin-Coated BeadsCaptured On A Fibril Mat

An AFP assay was run using the following steps: i) formation of asandwich immune complex on the surface of beads in suspension; ii)filtration of the beads onto a fibril mat supported on stainless steelfilter paper; and iii) detection of ECL from TAG1-labeled antibodies inthe immune complexes by scanning the fibril electrode to an oxidizingpotential. The assay was run using an AFP assay kit(Boehringer-Mannheim). This assay kit is designed for ECL-based assaysusing the Elecsys System (Boehringer-Mannheim); a system which capturesmagnetic beads on a platinum electrode by the application of a magneticfield. The assay kit included the following stock solutions: asuspension containing streptavidin-coated magnetic beads (M-280, DynalInc.), a biotin-labeled capture antibody (R-1), a TAG1-labeled secondaryantibody (R-2), and a series of calibrators containing AFP dissolved ina matrix designed to simulate serum.

To determine a calibration curve for the assay, the stock suspension ofstreptavidin-coated beads (0.017 mL containing ˜0.012 mg of beads) wascombined in a plastic tube with the stock solutions containing R-1(0.017 mL), R-2 (0.017 mL), and an AFP calibrator (0.010 mL). The tubewas vortexed and then gently shaken for 30 minutes at room temperature.The suspension was then filtered using gentle suction onto a fibril mat(mat thickness =0.030 mm) formed as described in Example 6.43. The matwas washed with ECL Assay Buffer (IGEN, Inc.). The ECL was then measuredas described in Example 6.26. Each calibrator was run in triplicate.FIG. 48 shows the background corrected ECL signal as a function of theconcentration of AFP.

6.45. A Particle-based ECL Assay For APP Using Streptavidin-CoatedSilica Beads Captured On A Fibril Mat

An AFP assay was run as described in Example 6.44 except thatstreptavidin-coated silica particles (Aerosil-200, prepared as describedin Example 6.42) were used instead of the magnetic beads supplied withthe AFP assay kit. To determine a calibration curve for the assay, thestock suspension of streptavidin-coated Aerosil-200 (0.010 mL containing0.001 mg of particles) was combined in a plastic tube with the stocksolutions containing R-1 (0.017 mL), R-2 (0.017 mL), and an AFPcalibrator (0.010 mL). The tube was vortexed and then gently shaken for30 minutes at room temperature. The suspension was then filtered usinggentle suction onto a fibril mat (mat thickness =40 mm) formed asdescribed in Example 6.43. The mat was washed with ECL Assay Buffer(IGEN, Inc.). The ECL was then measured as described in Example 6.26.Each calibrator was run in triplicate. FIG. 49 shows the backgroundcorrected ECL signal as a function of the concentration of AFP.

6.46. ECL Emitted From Fluorescent Dye-Labeled Latex Beads Captured On AFibril Mat Electrode

This example describes an experiment showing that fluorescent dyesincorporated into beads could be used as internal standards inbead-based ECL assays. Three types of fluorescent beads were purchasedfrom Polysciences Inc. The beads differed in the excitation and emissionwavelengths of the incorporated dyes: i) Catalog # 17685 (l_(ex)=273 nm,l_(ex)=340 nm). ii) Catalog # 19392 (l_(ex)=530 nm, l=590 nm). iii)Catalog # 17797 (l_(ex)=641 nm, l_(em)=740 nm). Fibril mats wereprepared as described in Example 6.43. The fluorescent beads (0.010 mg)were filtered on to the mats using gentle suction. The mats were washedwith ECL Assay Buffer (IGEN, Inc.) and the ECL measured as described inExample 6.26. Each bead was tested in triplicate. All three beadsemitted ECL under these conditions. The average integrated ECL signalsmeasured for Polyscience catalog # 17685, 19392, and 17797 beads were0.7 nA·s, 6.0 nA·s, and 2.3 nA·s, respectively.

6.47. An ECL Sandwich Immunoassay For AFP Using Biotin-StreptavidinCapture To Immobilize A Capture Antibody On A Gold Electrode

In this example, an antibody against alpha-fetoprotein (AFP) isimmobilized on a gold electrode for use in an immunoassay.

Glass slides (1 cm×1 cm×0.06 cm) were coated on one side with a thingold film by thermal evaporation. The gold film was formed by theevaporation of 3 nm of Ti as an adhesion layer followed by 100 nm of Au(99.99%). A self-assembed monolayer (SAM) was formed on the gold filmsby incubation of the slides in an ethanolic solution containingmercaptoundecanoic acid at a concentration of 1 mM for a period ofapproximately 10 hours. The carboxylic acid groups presented at thesurface of the SAM were activated by treatment for 10 minutes with 0.05mL of an aqueous solution containing1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-hydroxysuccinimide (NHS) at concentrations of 0.2 M and 50 mMrespectively. The surface was then washed briefly with water.Streptavidin was immobilized on the activated surface by treatment ofthe surface for 60 minutes with 0.05 mL of a solution containingstreptavidin at a concentration of 0.3 mg/mL in phosphate bufferedsaline (PBS). The surface was blocked by adding 0.08 mL of an aqueoussolution containing 1 M ethanolamine, pH 8.5. After incubating for 30minutes the chip was washed with HBS/p20 (10 mM HEPES, 0.15 M NaCl,0.005% Tween 20, pH 7.4).

The AFP assay kit (Boehringer-Mannheim) consists of a stock solution ofbiotin labeled primary antibody (0.007 mg/mL), a stock solution ofTAG1-labeled secondary antibody (0.012 mg/mL), and five calibratorsolutions in an artificial matrix designed to simulate human serum. Theassay conditions were not optimized for these reagents. To conduct anAFP assay, 0.020 ml of the primary antibody stock, 0.020 ml of thesecondary antibody stock, and 0.020 ml of a calibrator solution werecombined and applied to a slide that had been coated with streptavidin.The solution was incubated on the surface for 20 minutes. The surfacewas then washed with HBS/p20 (10 mM HEPES, 0.15 M NaCl, 0.005% Tween 20,pH 7.4).

ECL was measured on the surface of a slide by the following procedure:The slide was placed in an electrochemical cell. The cell contained anO-ring which defined the area of the gold surface which waselectrochemically excited (0.13 cm²). The cell also contained a Ptcounter electrode and a Ag/AgCl reference electrode as well as a meansto make electrical contact to the gold film. The cell was held in adefined position under a photomultiplier tube (PMT) to allow forreproducible detection of ECL emission. To excite ECL, the electrodeswere immersed in assay buffer (0.1 M tripropylamine, 0.2 M sodiumphosphate, 0.02% (w/v) Tween-20, pH 7.2) and the electrical potential atthe gold surface ramped from 0 V to 2 V (vs. Ag/AgCl) at a rate of0.2V/s. The photocurrent generated at the PMT was integrated over theduration of the scan to give an ECL signal in units of nA·s.

FIG. 51 shows a plot of the ECL signal vs. the concentration of AFP insolution.

6.48. The Detection Of Nucleic Acid Hybridization To A Probe ImmobilizedOn A Gold Electrode

The oligonucleotides used in this example are defined as follows:

-   SC1.2: 5′ ca gtt gtg tgc cac cta caa 3′ C6 Disulfide modifier-   SC2.3: 5′ ttg tag gtg gca cac aac tg 3′ C3 Amino Modifier-   SC4.1: 5′ TAG1-gaa-aat-gtg-ctg-acc-gga-cat-gaa-aat-gag 3′

The oligonucleotides were purchased from Oligos Etc. Inc. SC2.3 waslabeled with the TAG1 by reaction with a derivative of TAG1 presentingan NHS ester (NHS-TAG, IGEN Inc.). SC2.3 (130 nmole) in 0.100 ml of PBSpH 7.4 was combined with 0.400 ml of DMSO in a 0.5 mg vial ofTAG-NHS-ester. The solution was mixed and incubated overnight in thedark at room temperature. Following the overnight incubation thelabeling reaction was diluted with 1.380 ml of deionized water. Anaqueous solution containing sodium chloride at a concentration of 5 M(0.120 mL) was added followed by 0.120 mL of absolute ethanol. Thissolution was then incubated at −70° C. for at least 1 hour toprecipitate the product. The labeled oligonucleotide was centrifuged at5000×g for 10 minutes at 10° C. The resulting pellet was washed twicewith 0.5 mL of a 70% (v/v) solution of ethanol in water. The washedpellet was dried under vacuum and stored in the dark at −20° C. Themanufacturer of the SC4.1 oligonucleotide labeled the probe with TAG1during the oligonucleotide synthesis by reaction with a phosphoramiditederivative of TAG1 (IGEN, Inc.).

Glass slides (1 cm×1 cm×0.06 cm) were coated on one side with a thingold film by thermal evaporation. The gold films were formed by theevaporation of 4 nm of Ti as an adhesion layer followed by 200 nm of Au(99.99%). Wells were formed above the gold films by sealing the slides,with O-rings, against holes drilled through a block of plastic. TheO-rings defined the area of the gold films in contact with solution(0.25 cm²). The oligonucleotide SC1.2 was immobilized on the surface ofthe gold films by adding 0.050 mL of a solution containing 0.010 mg ofthe oligonucleotide (in 10 mM ammonium acetate, pH 6.0) to each well.The immobilization was allowed to proceed overnight in a darkdesiccator, during which time the solution containing theoligonucleotide evaporated to dryness. Excess reagents were then removedby several washes with deionized water.

Prehybridization of the oligonucleotides on the surface of the goldslide was accomplished by the addition of 0.050 mL of a solutioncontaining the components of SSC solution (1×), the components ofDenhardts Solution(1×), yeast tRNA (0.100 mg/mL), and sonicated herringsperm DNA (0.050 mg/mL). The slides were then shaken vigorously for 30minutes at room temperature. Following the prehybridization step, theslides were washed with SSC and hybridized with calibration solutionscontaining (1=10¹²) molecules of TAG1-labeled SC2.3 (an oligonucleotidecomplementary to the sequence immobilized on the surface), orTAG1-labeled SC4.1 (a non-complementary probe used as a negative controlto test for non-specific binding). The TAG1-labeled probes were appliedas solutions in 0.050 ml of the solution used for prehybridization. Thehybridization was allowed to proceed for 2 hours at room temperaturewith vigorous shaking. The gold slides were then washed three times with1×SSC containing 0.1% (w/v) SDS, incubated with the same buffer for 30minutes at room temperature and rinsed with ECL assay buffer (IGEN,Inc.). ECL was excited from the surface of the gold film as described inExample 6.47. ECL was also excited from a surface that was not exposedto a TAG1 -labeled probe in order to measure the magnitude of thebackground signal. The value of the background corrected signal from thecomplementary probe was 3.3×10³ nA·s, showing that the TAG1-labeledprobe hybridized to the surface and could be detected by ECL. The valueof the background corrected signal from the non-complementary probe was−2.2×10¹ nA·s, showing that the binding of the non-complementary probewas specific and that the non-specific binding was low.

6.49. Preparation Of Sheets Of A Composite Electrode Containing FibrilsAnd EVA

A composite electrode was prepared by compounding fibrils with a polymerand compression molding the compounded material into a sheet. Fibrilswere compounded into EVA by using a Brabender Plasticorder with a twinscrew metering head at a temperature of 180 degrees centigrade and aspeed of 100 r.p.m. 9.45 grams of fibrils were dry blended with 25.55grams of EVA (Quantum Chemical, Microthene, FS-532). The blendedmaterials were added to the mixing head over a period of 1 minute toallow the material to melt. Mixing was continued for an additional 5minutes and then the composite was removed from the mixing head andallowed to cool. To prepare sheets of the composite for use as anelectrode, a 2 gram piece of the compounded material was assembled intoa sandwich between two polished stainless steel plates (triple platedferrotype plates, Testrite Company) and the assembly was placed betweenheated platens set at 180 degrees Centigrade in a hydraulic press(Carver). After allowing time for the material to be heated, thecomposite was pressed into a flat sheet at 10000 pounds total pressure.The.assembly was then removed from the press and allowed to cool to roomtemperature. The assembly was separated and a flat disk with nominalthickness of 20 mils was removed.

6.50. Oxidation Of Fibril-Polymer Composites By Chromic Acid

Composites of carbon fibrils (27% by weight) with ethyl vinylacetate(fibril-EVA) and carbon fibrils with polyethylene (fibril-PE) were used.The composites were obtained as 3″ disks that were approximately 1 mmthick. Both fibril-EVA and fibril-PE composites were oxidized byfloating the disks on a solution containing chromic acid (CrO₃, H₂ O andH₂SO₄ (29/42/29; w/w/w)), at room temperature for 1 hour. After reactionwith the chromic acid solution, the oxidized composites were then washed4-5 times with deionized water, soaked for at least 5 minutes indeionized water and dried in air for 1 hour.

6.51. Derivatization Of A Fibril-Polymer Composite With A Mixture ofSulfuric And Nitric Acids

A composite of EVA and carbon fibrils (in the form of a 3″ diameter flatdisk) was treated with a mixture of sulfuric acid and nitric acid with1:1 ratio (12 mL) for 3 hours. The treated composite was washed withwater. To the treated composite in a mixture of water (150 mL) andammonium hydroxide (30%, 150 mL) was added sodium dithionite (10 grams).The reaction mixture was refluxed for 2 hours. The composite was washedextensively with water.

6.52. Preparation Of A Fibril Composite Electrode With Exposed HydroxylGroups

Hydroxyl groups were exposed on fibril-EVA composites by hydrolysis ofacetate groups at and near the surface of the composite. Discs (3″diameter, 0.01″ thickness) of the composite material (27% CC fibrils inEVA, by weight) were immersed in 100 mL of a 2 M solution of NaOH for17-20 hours at room temperature. This treatment leads to the exposure ofhydroxyl groups on both sides of the composite. The composite was washedwith water and methanol and then allowed to dry in air.

6.53. Immobilization Of streptavidin On Oxidized Fibril-PolymerComposites

EVA-fibril composites were oxidized as described in example 6.50 or byexposure to an oxygen plasma. The oxidized 3″ disc composites were driedunder vacuum pump for 1 hour and soaked overnight (with shaking) in 25ml of dichloromethane containing 0.1 M EDC(1-ethyl-3-(3-dimethylaminopropyl(carbodiimide) and 0.1 MN-hydroxysuccinamide. The NHS activated composites were washed withdichloromethane, methanol, deionized water and methanol, then allowed todry at room temperature.

For immobilization of streptavidin, the NHS activated composites wererinsed with deionized water and floated on 6 mL of streptavidin solutionso that the NHS-ester activated surface faced down. The streptavidinsolution was prepared in PBS-1 (0.1 M sodium phosphate, 0.15 M sodiumchloride, pH=7.8) in a concentration of 0.7 mg/ml. The composite wasshaken for 3 hours in the streptavidin solution and washed by shaking in20 ml of PBS-1 containing 0.1% Triton for 30 minutes (one time) and in20 ml of PBS-1 for 30 minutes (5 times). The streptavidin loaded EVAcomposites were stored in PBS-1 at 4° C.

6.54. Immobilization Of Proteins On Fibril-Polymer Composites Via SMCCActivation

A 3″ diameter disc of EVA-fibril composite that had been treated with amixture of nitric and sulfuric acids (as in Example 6.51) was placed in15 mL of sodium phosphate buffer (0.1 M, pH 7.5).Sulfosuccinimididyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate(sulfo-SMCC, 10 mg) was added and the reaction mixture was incubated for3 hours at room temperature. The reaction was stopped and the compositewas washed with sodium phosphate buffer. To a solution of streptavidin(4.5 mg) in 0.1 M sodium phosphate, 20 mM EDTA, pH 7.5 (300 mL), wasadded Traut's reagent (19.8 mL, 2.8 mg/mL). The reaction mixture wasincubated at room temperature for 1 hour and 30 minutes. Thesulfhydryl-labeled Streptavidin was purified using a PD-10 (Pharmacia)disposable size exclusion column (collected in a 3.5 mL fraction). TheSMCC-treated composite (1 inch, derived via nitration/reduction of EVACC composite) was placed in the solution containing sulfhydryl-labeledstreptavidin (3.5 mL) and incubated with shaking for overnight at 4° C.The resulting streptavidin-coated composite was washed (5×20 minutes) byshaking in 0.1 M sodium phosphate, pH 7.5, containing 1% Triton X-100.

6.55. Immobilization of Streptavidin On A Composite Electrode PresentingExposed Hydroxyl Groups

Streptavidin was immobilized on a fibril-EVA composite that presentedhydroxyl groups at and near the surface. The protein was immobilizedafter activation of the hydroxyl groups with carbonyldiimidazole (CDI).Fibril-EVA composite electrodes that had been treated with NaOH toexpose hydroxyl groups (by the procedure described in Example 6.52) weredried under vacuum. The dry composite was immersed in 50 mL of anhydrousmethylene chloride in a dry glass jar. To the solution was added 100 mg(0.6 mmol) of 1,1′-carbonyldiimidazole (CDI) and the jar was gentlyrocked for 1 h at room temperature. The composite was then washed withmethylene chloride and methanol and then allowed to dry in air. The CDIactivated composite was cut (using a punch) into 3/16″ discs (96 total).Each disc was placed at the bottom of the well of a 96 well microtiterplate. Streptavidin (100 mL of a 0.1 mg/mL solution in 0.2 M sodiumbicarbonate, pH 8.5) was then added to each well. The immobilization wasallowed to proceed for 18-20 h at 4° C. The discs were washed threetimes by soaking in 100 ml portions of 50 mM sodium phosphate, pH 7.5and stored in 100 mL of 50 mM sodium phosphate, pH 7.2 until used.

6.56. Assay For AFP On A Composite Electrode Of EVA And Fibrils

The AFP assay was performed using an EVA-fibril composite electrode withstreptavidin immobilized on it (prepared as described in Example 6.53).The assay was carried out in a ninety-six well plate that was preblockedwith 1% BSA solution (1% BSA and 0.3% Tween 20 in 0.1 M sodiumphosphate, pH=6.8) and rinsed once with 0.3% Tween 20 in 0.1 M sodiumphosphate, pH=6.8, prior to the assay. Forty eight discs with diametersof 3/16″ were punched from a 3′ diameter EVA-SA composite and placed inthe ninety-six well plate with the treated surfaces up. Fiftymicroliters of biotinylated AFP antibody were added to the wells thatcontained composites. The plate was then shaken at room temperature for30 minutes. The composites were rinsed twice with 0.1 mL of PBS-1 andshaken for 1 hour at room temperature with the mixture of 0.05 mL ofcalibrators and 0.05 mL of TAG1 labeled antibody. At the end of thereaction, composites were rinsed with 0.150 mL of ECL assay buffer andstored in protein buffer (3% BSA, 3% Tween 20, 25 mM sodium chloride and0.1 M sodium phosphate, pH=7.3) until the ECL was measured. We usedeight AFP calibrators ranging from 0.56 to 7950 IU/ml. Each calibratorwas assayed in six replicates. FIG. 53 shows the results.

6.57. ECL Assay For TSH Using A Composite Electrode Of EVA And Fibrils

The assay was conducted using 3/16″ diameter streptavidin-coated discsof EVA-fibril composite (prepared as described in Example 6.55). Theassay reagents were part of a TSH assay kit (IGEN, Inc.) and included abiotin-labeled anti-TSH antibody and a TAG1-labeled anti-TSH antibody,each dissolved in a buffer (TSH Assay Diluent). The discs were placedinto individual wells of a 96-well microtiter plate. The biotin-labeledantibody (0.05 mg, 0.012 mg/mL) was added to each well and the plate wasgently shaken for 30 minutes at 37° C. After washing the discs with TSHAssay Diluent, solutions containing the TAG1-labeled antibody (0.025 mL,600 ng/mL) and varying amounts of TSH were added and the plate shakenanother 60 minutes at 37° C. The discs were washed with and stored inTSH Assay Diluent until their analysis by ECL.

6.58. DNA Hybridization Assay On A Fibril-Polymer Composite

This example describes an assay for a sequence of DNA. The assayinvolves the formation of a sandwich complex on a streptavidin-coatedcomposite electrode which includes a biotin-labeled capture probe (SC5),an analyte (SC3.1) and a TAG1-labeled probe (SC4.1). SC3.1 and SC4.1 aredescribed in Example 6.48. SC5 was purchased from Oligos, Etc., Inc. andhas the sequence 5′-ca gtt gtg tgc cac cta caa gca tta cgg act agt catggt tca cag agg-3′-biotin. Oxidized fibril-EVA composites were activatedwith EDC and NHS as described in Example 6.53. Wells were defined above⅜″ discs of the composite by sealing the discs, with O-rings, againstholes drilled through a block of plastic. The O-rings defined the areaof the composite (0.25 cm²) in contact with solution placed in thewells. Streptavidin was immobilized on the discs by adding 0.05 mL of a0.5 mg/mL solution of Streptavidin (in PBS) to each well. The reactionwas allowed to proceed for 3 hours at room temperature while shaking theapparatus. The discs were washed twice with PBS, once with PBScontaining 0.10% (w/v) Triton x-100 and twice more with PBS.

To capture the biotin labeled oligo we added 10¹³ molecules of SC5 in0.05 mL of ECL Assay Buffer (IGEN, Inc.) and incubated for 2 hours atroom temperature with vigorous shaking. Excess SC5 was removed bywashing with PBS, PBS containing 0.10% (w/v) Triton x-100, and ECL AssayBuffer. SC4.1 (10¹² molecules in 0.025 mL) and SC3.1 (varying amounts in0.025 mL) in ECL Assay Buffer were then added. The hybridizationreactions were allowed to proceed for 4 hours. Excess reagents were thenremoved by washing with PBS, PBS containing 0.10% (w/v) Triton X-100 andECL Assay Buffer. The discs were then placed in an ECL apparatus and theECL signal was measured. FIG. 54 shows the ECL signal as a function ofthe amount of SC3.1.

6.59. Measurement Of The Surface Area Of A Fibril-Composite Electrode

The amount of fibrils protruding from the surface of a compositeelectrode can be estimated from measurements of the double layercapacitance. Electrodes were prepared by punching out 0.25 inch diameterdisks of composite and making electrical contact to one surface byattaching a copper wire with electrical paint. Any exposed copper wirewas sealed in epoxy. The electrodes were used to record cyclicvoltammograms in argon-purged 0.5M K₂SO₄ at several potential scan rates(e.g. 5, 10 and 25 mV/second) between −0.2V vs, Ag/AgCl and +0.8V vs.Ag/AgCl. The double layer charging current, I_(dl) was measured in thebroad, flat region of the cyclic voltammograms, typically at +0.25V vs.Ag/AgCl. The slope of plots at I_(dl) vs. scan rate is taken as thedouble layer capacitance C_(dl). Using an average value of 10 μF/cm² offibril surface area, and a fibril surface area of 200 M²/gram, theamount of fibrils exposed to the electrolyte can be estimated.

6.60. Preparation Of NHS Ester-Functionalized Fibrils

N-Hydroxysuccinimide (NHS) (0.35 g) and1-ethyl-3-(3-dimethylaminopropyl) cardodiimide (EDC) (0.60 mg) wereadded to a suspension containing 220 mg of carboxylated fibrils(provided by Hyperion Catalysts Inc.) in 25 mL of dioxane and themixture was sonified (Sonifier 250, Branson Ultrasonics) for 5 minutesand stirred at room temperature overnight. The reaction was stopped byvacuum filtration of the reactants from the fibrils in a sintered glassfunnel. The fibrils were washed with dioxane (3×15 mL) and methanol(extensively) then dried under vacuum to yield 220 mg NHSester-activated fibrils.

6.61. Conjugation Of Streptavidin To NHS Ester Fibrils

NHS Ester-modified fibrils (2.1 mg, prepared as described in Example6.60) were sonified (Sonifier 250, Branson Ultrasonics) in 400 μL PBS-1buffer (0.1 M sodium phosphate, 0.15 M sodium chloride, pH=7.8) for 5minutes at the lowest power setting. A solution of streptavidin (2.4 mgin 150 μL PBS-1) was mixed with the dispersed fibril suspension and themixture (650 μL) was gently shaken for 3 hours at room temperature. Thefibrils were washed by multiple cycles of repetitive centrifugation, andresuspension using the following buffers in series: 0.1 M sodiumphosphate containing 1% Triton X-100 (1 time), PBS-1 (two times), 0.1 Msodium phosphate containing 0.1% Triton X-100 (1 time), and PBS-1 (4times). Streptavidin loaded fibrils were stored at 4° C. in PBS-1.

6.62. Fabrication Of A Ultra Thin Fibril Mat (UTFM) On A Nylon MembraneFilter

An aqueous suspension containing CC fibrils at a concentration of 0.1mg/mL was prepared by diluting a stock suspension of the CC fibrils (1mg/mL in water) into an aqueous solution of Triton X-100 (0.2% (w/v)).The CC fibrils were finely dispersed with a probe sonicator (Sonifier250, Branson Ultrasonics) by sonicating for 5 min using a duty cycle of30% and setting the output control to a value of 3. A further dilutionof the sonified suspension to a concentration of 0.01 mg/mL was thencarried out by diluting 4 mL of the suspension up to a volume of 40 mLwith the aqueous solution of Triton X-100.

A UTFM was prepared on a nylon membrane (0.45 mm pore size, 47 mmdiameter) by vacuum filtration (see FIG. 24 for an example of anapparatus for filtration of fibril mats). The finely dispersed fibrilswere filtered onto the nylon membrane in four aliquots of 4 mL eachusing a vacuum of approximately 26 in. of Hg. Vacuum filtration wascontinued until only a trace of liquid remained unfiltered above themembrane (by visual observation). The mat was then removed from thefiltration apparatus, compressed between two pieces of clean, dry filterpaper, and allowed to dry flat in an oven for approximately 10-15minutes at 60° C.

The volumes used could be scaled accordingly for electrodes of differentareas and/or thickness.

6.63. A Nucleic Acid Hybridization Assay On A Bilaver Ultra Thin FibrilMat Electrode

Streptavidin was covalently immobilized on dispersed CC carbon fibrilsas described in Example 6.61. A total of 100 μg of these fibrils weresuspended in a solution containing BSA at a concentration of 1 mg/mL(w/v) to block unoccupied sites on the fibrils. The blocked fibrils werethen centrifuged and resuspended in 1 mL of deionized water. Thissuspension was redispersed by sonification (Sonifier 250, BransonUltrasonics) for 5 minutes.

An ultra-thin fibril mat (UTFM) was prepared as described in Example6.62. After the UTFM was prepared, the second layer was formed by thefiltration of 17 μL of the suspension of streptavidin-fibrils onto thefirst layer under the same conditions as used in Example 6.62.

To conduct the DNA hybridization assay, we used a “capture”oligonucleotide (28 base pairs, biotinylated at the 5′ position) thatwas allowed to hybridize to a complementary “TAGged” oligonucleotide (28base pairs, end labeled with TAG1-NHS Ester). The assay was prepared bythe following steps: i) calibration solutions were prepared thatcontained variable concentrations of the biotin-labeled oligo and aconstant excess (10¹² molecules) of the TAG1-labeled oligo; ii) thecalibration solutions (50 AL) were filtered through ultra-thin fibrilmats (one UTFM per solution) at a flow velocity of 50 μm/sec to allowcapture of the biotinylated complex onto the streptavidin-coated fibrilsin the fibril mat; iii) the UTFM was washed with 50 AL of ECL AssayBuffer (IGEN, Inc.) to remove unbound reagents, and iv) the UTFMs (andthe attached oligonucleotide complexes) were transferred to ameasurement cell (FIG. 34) and ECL was measured. FIG. 55 shows that theassay measured the TAG1-labeled oligonucleotide with high sensitivityand produced a linear response over a wide dynamic range.

6.64. A Sandwich Immunoassay For AFP On A Bilayer Ultra Thin Fibril MatElectrode

A suspension of streptavidin-coated (and BSA-blocked) fibrils wasprepared as described in Example 6.61. The suspension was diluted in ECLAssay Buffer (IGEN, Inc.) to give a stock suspension with aconcentration of fibrils of 7 mg/mL. The solution was placed in an icewater bath (to prevent denaturing of the streptavidin) and the fibrilswere redispersed by sonication (Sonifier 250, Branson Ultrasonics) for 5minutes using a duty cycle of 20% and setting the output control to avalue of 1.5.

A vacuum filtration apparatus was used to prepare bilayer UTFMelectrodes on nylon filter membranes (0.45 mm pore size). The filtrationapparatus defined a ⅛″ diameter area on the membrane through whichfiltration occurred. A layer of underivatized fibrils was formed byfiltering (using suction) 87.5 μL of a finely dispersed suspensioncontaining underivatized fibrils at a concentration of 0.01 mg/mL(prepared as described in Example 6.62). A second layer was formed byfiltering 50 μL of the stock suspension of streptavidin-coated fibrilsonto the layer of underivatized fibrils.

The AFP assay reagents (Elecsys, Boehringer-Mannheim) were pre-mixedprior to filtration by combining 50 μL of the stock solution of thebiotinylated AFP antibody, 50 μL of the stock solution of theTAG1-labeled AFP antibody, and 10 μL of one of several stock solutionscontaining known concentrations of AFP. The combined solutions were thenfiltered by suction at a controlled flow velocity using a vacuumpressure of 5 in. of Hg. Each sample took between 30-50 minutes tofilter. The mats were then washed with 150 μL of ECL Assay Buffer (IGEN,Inc.), removed and allowed to dry. The mats were transferred to ameasurement cell (FIG. 34) and ECL was measured. FIG. 56 shows the ECLsignal as a function of the concentration of AFP in the calibrationsolution.

6.65. Forming Conductive Films Of Gold on Non-conductive FilterMembranes

Nylon membrane filters from Whatman (0.45 μm pore size) were coated withsputtered gold using a Balzers MED 010 Minideposition System. Thedeposition was carried out using an argon plasma (at a pressure of5×10⁻² mbar) and a discharge current of 100 mA.

6.66. A Sandwich Immunoassay For AFP On An Ultra Thin Fibril MatElectrode Formed On A Gold-Coated Nylon Filter Membrane

A suspension of streptavidin-coated (and BSA-blocked) fibrils wasprepared as described in Example 6.61. The suspension was diluted in ECLAssay Buffer (IGEN, Inc.) to give a stock suspension with aconcentration of fibrils of 28 mg/mL. The solution was placed in an icewater bath (to prevent denaturing of the streptavidin) and the fibrilswere redispersed by sonication (Sonifier 250, Branson Ultrasonics) for 5minutes using a duty cycle of 20% and setting the output control to avalue of 1.5.

A total of thirty-six 5/16″ diameter disks were punched out of Aconductive Au-coated nylon filter membranes (100 nm gold film, preparedas described in Example 6.65) was cut (using a hole punch) to form 5/16″diameter discs. The disks were placed into the wells of a multiplesample filtration apparatus, which allows for as many as ninety-sixsamples at a time to be filtered under a controlled vacuum pressure.This apparatus filters samples onto a 3/16″ diameter circular region oneach disc. Single layer UTFMs were formed by filtering (using a vacuumof 26 in. of Hg) 82.5 μL of the stock solution of streptavidin-coatedfibrils onto the discs. The AFP assay reagents (Elecsys,Boehringer-Mannheim) were pre-mixed prior to filtration by combining 50μL of the stock solution of the biotinylated AFP antibody, 50 μL of thestock solution of the TAG1-labeled AFP antibody, and 10 μL of one ofseveral stock solutions containing known concentrations of AFP. Thecombined solutions were then filtered by suction at a controlled flowvelocity using a vacuum pressure of 5 in. of Hg. Each sample tookbetween 30 minutes to filter. Each mat was then washed with 150 μL ofassay buffer, then removed and stored in a protein buffer which contains3.0% BSA, 3.0% Tween-20, 25 mM NaCl, and 100 mM NaH₂PO₄. The mats weretransferred to a measurement cell (FIG. 34) and ECL was measured. FIG.57 shows the ECL signal as a function of the concentration of AFP in thecalibration solution.

6.67. AFP Assay On Two Different Electrodes:

Voltammetric Resolution Of Signal And Background

The AFP assay was developed on two composite electrodes. Both electrodeswere prepared from an ethylene vinyl acetate (EVA) copolymer containing27% CC-fibrils by weight. One electrode (the “hydrolyzed EVA” electrode)was treated with potassium hydroxide, activated with CDI, and exposed tostreptavidin (as in Example 6.55). The other electrode (the “chromicacid EVA” electrode) was treated with chromic acid, treated with NHS andEDC and exposed to streptavidin (as in Example 6.53).

Sandwich complexes containing a biotin-labeled anti-AFP antibody, aTAG1-labeled anti-AFP antibody and AFP were captured on 3/16″ discs ofthe two electrodes by the procedures described in Example 6.56.

Each electrode was installed in an ECL test cell (FIG. 34) with a gasket(⅛″ id., ⅞″ od., and 0.017″ thick) which defined the active area of theelectrode (⅛″ diameter). The test cell consisted of a black Delrin bodythat contained a three electrode electrochemical cell and an opticalwindow parallel with the working electrode. The electrodes were a 3MAg/AgCl reference electrode, a platinum mesh counter electrode, and thefibril-EVA composite as working electrode. The test cell was filled withca. 1 mL of Assay Buffer and placed in a light-tight box that was heatedto ca. 33° C. with a prototype electrical heater. The test cell opticalwindow was placed in front of a PMT and the box was closed. The PMT waspowered at 900 V with a Pacific Instruments Model 126 Photometer runningin analog mode with a 1 second time constant filter using several of thephotocurrent ranges (i.e. 10 nA/V, 30 nA/V, 100 nA/V, 300 nA/V, and 1000nA/V). The electrochemical cell was controlled by an EG&G PARC Model 175Universal Programmer and an EG&G PAR Model 173 Potentiostat/Galvanostat.Following a 100 s delay at 0 V vs. 3M Ag/AgCl the potential was swept at100 mV/s from 0 V (E₀), to a lower limit of −0.8 V (E₁), an upper limitof 2.3 V (E₂) and an ending potential of 0 V (E₃). The current range wasset at 1 mA/V. The air temperature around the test cell was measuredwith a Cole Parmer Thermistor Thermometer and probe. All analog data(temperature, light, applied potential, and current) was digitized at 10Hz with a Computer Boards Inc. CIO-DAS1602/16 A/D board controlled byHEM Data Corp. Snap-Master for Windows J in a Pyramid 100 MHz Pentiumcomputer. From the raw data, plots of ECL and voltammetric current vs.applied potential were prepared and several data were calculatedincluding: maximum anodic current, ECL peak potential (applied potentialat the ECL peak), mean ECL dark current (mean light between startingpotential and 0.5 V), and dark corrected integrated ECL (the differencebetween the average ECL over a given potential range and the mean darkall of which was then multiplied by the duration in seconds of the givenpotential range).

The voltammetry from the AFP assays are dependent upon the electrodeused. The voltammograms for the AFP assay on hydrolyzed EVA consist ofan irreversible oxidative wave starting at 1.4 V and having an anodicpeak at ca. 1.8 V. The maximum anodic current occurs at 2.3 V. Littlecurrent is passed between −0.8 V and 1.4 V. The voltammograms fromchromic acid treated EVA consist of a very broad irreversible oxidativewave starting at 0.7 V and having several undefined anodic peaks (ca.1.1, 1.5, and 2.0 V) and a maximum anodic current at 2.3 V. Littlecurrent was passed between −0.8 and 0.7 V.

The hydrolyzed EVA yielded ECL traces consisting of a peak at with asmall amount of tailing to the high potential side. The peak potentialsshifted slightly from 1.85 V for blank signals to 1.75 V for analytesignals. The chromic acid treated EVA yielded two closely spaced peaks,both of which scaled with analyte concentration. These peaks were at ca.1.6 V and ca. 1.25 V. At low analyte concentrations the peak at 1.6 Vwas dominant while at higher analyte concentrations the peak at 1.25 Vwas dominant. The chromic acid treatment increased the peak-to-peakresolution from ca. 100 mV (hydrolyzed EVA) to ca. 350 mV. This peakshift was sufficient to allow the analysis of the first peak which hasbeen shown to be more sensitive to analyte and to reduce the amount ofthe blank signal used in the analysis.

In similar experiments, we compared the ECL traces for AFP calibrator 1(predominantly ECL assay buffer, FIG. 60) and AFP calibrator 3 (FIG.61). In FIG. 60, the ECL signal corresponding to assay buffer appears asa peak with a maximum at 1.5V. In FIG. 61, the ECL signal for assaybuffer is also at 1.5V; the additional peak at 1.0V corresponds to thesignal from the analyte in a sandwich complex that includes aTAG1-labeled complementary antibody.

6.68. Preparation of Extruded Sheets of Fibril-EVA Composites

We blended 270 grams of carbon nanotubes (HCI Fibrils, CC grade) and 730grams of ethyl vinylacetate (EVA, Quantum Microthene FE530) in aHenschel lab mixer for 2 minutes. The nanotube-EVA blend was vacuumtransfered to a sealed hopper, compounded on a Buss PR 46 kneader at180° C., and fed into a Buss cross head pelletizer. The pellets weredried in a Gala dryer. This process gave a composite containing 27%fibrils by weight.

Prior to extrusion, pellets of compounded nanotube-EVA composite weredried at 80° C. for at least 12 hours. The dried pellets were starve fedinto a Brabender PL2000 Plasti-Corder (a ¾″ 25:1 L/D single screwextruder equipped with a 2:1 compression ratio screw and a 6″ flex-lipdie). The temperature of the three heating zones of the extruder and thedie was 245° C. The output die of the extruder produced a continuousribbon (˜15 cm wide, ˜1 mm thick) of nanotube-EVA composite that wascollected on a conveyor belt at ambient temperature with negligible drawdown. The surface of the composite that did not contact the conveyorbelt was used for all further experiments.

6.69. Use of Oxygen Plasma To Chemically Modify Fibril Composites ForECL Immunoassays

Composites of carbon fibrils and EVA (27% fibrils by weight) wereexposed to a plasma formed from oxygen (O₂) gas. The composites wereexposed to the plasma for 10 minutes at 2000 W (20 kwmin). To introduceN-hydroxysuccinimide-ester functional groups (NHS-ester), theplasma-treated fibril-EVA composites (126 cm²) were reacted withN-hydroxysuccinimide (700 mg, from Aldrich 13067-2) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.6 g, fromAldrich 16146-2) in anhydrous methylene chloride (50 mL) for 2 hours atroom temperature, then rinsed with methanol and water, and blown drywith a stream of argon gas. The NHS-ester derivatized composites wereincubated with streptavidin (5 mg, from Pierce 21125) in PBS-1 buffer(0.1 M sodium phosphate, 0.15 M sodium chloride, pH=7.8, 50 mL) for 4hours at room temperature. Then the composites were washed with 1%Triton X-100 in PBS-1 for 30 minutes shaking and three times with PBS-1for 30 minutes shaking each.

6.70. Use of oxygen Plasma (40 KWmin) Followed by NH₃/N₂ Plasma (4:1,120 KWmin) To Chemically Modify Fibril Composites

Composites (123 cm²) of carbon fibrils and EVA (27% fibrils by weight)were first exposed to a plasma formed from oxygen (O₂) gas. Thecomposites were exposed to the plasma for 20 minutes at 2000 W (40kwmin). Then the composites were exposed to a plasma formed from amixture of ammonia (NH₃) gas and nitrogen (N₂) gas (4:1 ratio of thegases respectively). The composites were exposed to this plasma for 60minutes at 2000W (120 kwmin). To form a maleimide functional group, theplasma-treated fibril-EVA composites were incubated with Sulfo-SMCC (35mg, from Pierce: 22322) in PBS-1 (50 ml) for 3 hours with shaking atroom temperature. Then the composites were washed once with water (60 mlwith 10 minutes shaking) and three times with PBS-1 (60 ml with 10minutes shaking each).

To prepare IgG for immobilization on the activated composites,dithiothreitol (5 mg, from Sigma D-9779) was added to anti-AFP IgG (fromBoehringer Mannhem) solution (5.5 mg in 500 μl PBS-1 buffer). Themixture was incubated for 30 minutes (with rotating to mix) at roomtemperature. This procedure exposed thiol groups on the IgG(“IgG-(SH)_(n)”) . The “IgG-(SH)_(n)” was purified by columnchromatography, and then diluted into 20 mM EDTA PBS-1 buffer.

The maleimide-activated composite (60 cm²) was incubated with thepurified “IgG-(SH)_(n)” solution (5.5 mg “IgG-(SH)_(n)” in 30 ml of 20mM EDTA PBS-1 buffer) for 2.5 hours at room temperature. The compositewas washed once with 1% Triton X-100 (60 ml) for 20 minutes with shakingand three times with PBS-1 for 20 minutes each with shaking. Theanti-AFP-IgG composite was stored in PBS-1.

An AFP assay was performed on the anti-AFP-IgG composites. A 96-wellplate was precoated with BSA. Individual disks ( 3/16″ diameter) of thecomposites were punched out by hand with a metal punch. Each disk wasincubated with a solution containing TAG1-labeled IgG (100 μL) and asample containing a known amount of AFP (20 μL) for 60 minutes at roomtemperature. After incubation, the disks were rinsed three times withPBS-1 (200 μL) and stored in BSA assay diluent (100 μL). FIG. 62 shows aplot of the log of the difference between the ECL signal for samplescontaining AFP and a sample that contained no AFP (the backgroundsample) as a function of the log of the concentration of AFP in thesample.

6.71. Adsorption of Proteins on Composite Electrodes that were Oxidizedin a Water/Argon Plasma

Fibril-EVA composites electrodes (prepared as described in Example 6.68)were treated with a plasma formed from water-saturated argon in anAdvanced Plasma Systems Series C plasma reactor (1 hour, 2000W, 300mtorr). A composite (˜80 cm²) was placed in 20 mL of a solutioncontaining an anti-AFP monoclonal (Beohringer-Mannheim) at aconcentration of 0.2 mg/mL in 100 mM phosphate, pH 7.5 and incubatedwith gentle agitation for 2 hours at room temperature. The composite waswashed with 100 mM phosphate, pH 7.5 and stored in the same solution at−4 C. The amount of adsorbed antibody on the disk was determined,through the use of radiolabeling, to be 2.9 pmol.

Sandwich immunoassays for AFP were conducted on these disks. Samples (20uL) containing AFP were combined with 50 uL of a solution containing aTAG1-labeled secondary antibody (Boehringer-Mannheim) at a concentrationof 12 ug/mL. The surface was treated with this mixture and then washedwith phosphate buffer. ECL was measured by contacting the compositeelectrode with a solution containing tripropylamine (Assay Buffer, IGEN)and scanning the potential at the composite electrode from 0 V to −0.8 Vto 2.3 V (vs. Ag/AgCl) at a scan rate of 0.1 V/s. The ECL signalsobserved for samples with known amounts of AFP are shown in FIG. 63.

6.72. AFP Assays Using Fibril-EVA Composite Electrodes Containing 15 %Fibrils by Weight

Pellets of fibril-EVA composites containing a 27% fibrils by weight werecompounded with additional EVA to produce a fibril-EVA compositecontaining 15% fibrils by weight. This composite was extruded intosheets by a procedure analogous to that described in Example 6.68. Thecomposite was treated with an Argon/Water plasma and coated with ananti-AFP antibody as described in Example 6.71. Studies using aradiolabeled antibody showed that these composites containing only 15%fibrils by weight adsorbed more antibody (3.13 pmol) than othercomposites containing 27% fibrils by weight (2.9 pmol, see Example6.71). The antibody-coated composites were used in AFP assays asdescribed in Example 6.71. The ECL signals that were observed forsamples containing known amounts of AFP are shown in FIG. 64. Thecomposites containing only 15% fibrils by weight gave slightly higherECL signals (FIG. 64) than composites containing 27% fibrils by weight(FIG. 63, Example 6.71).

6.73. Adsorption of Proteins on a Plasma-Grafted Layer of Octadecylamineon a Plasma-Oxidized Composite

Fibril-EVA composites (prepared as described in Example 6.68) weresoaked for 2 hours in a solution containing octadecyl amine at aconcentration of 1 mg/mL in chloroform. The composite was dried in air(weight was applied at the edges to keep the sheet of composite flatduring the drying process). The composite was then treated with anoxygen plasma in an Advanced Plasma Systems Series C plasma reactor (30min., 2000 W, 300 mtorr). The composite was then coated with avidin bysoaking the material in a solution containing avidin (1.25 mg/mL) in 5mM phosphate, pH 7.5. Excess avidin was removed by washing withphosphate buffered saline. Radioisotope experiments using ¹²⁵I-labeledstreptavidin and ¹²⁵I and biotin-labeled rabbit IgG showed that weimmobilized 30-43 pmols of streptavidin and that the streptavidin wascapable of binding >2.2 pmol of biotin-labeled antibody.

Sandwich immunoassays for AFP were conducted on these disks. The surfacewas treated with 100 uL of a solution containing a biotin-labeledanti-AFP antibody (7 ug/mL, Boehringer-Mannheim) for 30 min. withshaking and then washed with phosphate buffered saline. A 20 uL samplewas combined with 100 uL of a solution containing a TAG1-labeledsecondary antibody (Boehringer-Mannheim) at a concentration of 12 ug/mL.The surface was treated with this mixture for 60 min while shaking andthen washed with Assay Buffer (IGEN). ECL was conducted by contactingthe composite electrode with a solution containing tripropylamine (AssayBuffer, IGEN) and scanning the potential at the composite electrode from0 V to −0.8 V to 2.3 V (vs. Ag/AgCl) at a scan rate of 0.1 V/s. The ECLsignals observed for samples with known amounts of AFP are shown in FIG.65.

6.74. Coupling of Proteins to Functional Groups Grafted ontoPlasma-Treated Composites

Fibril-EVA composites (prepared as described in Example 6.68) weretreated with an argon plasma in an Advanced Plasma Systems Series Cplasma reactor (1000 W, 3 min., 300 mtorr). The composites were removedfrom the reactor and immediately placed in oxygen-free solutionscontaining 4% (w/v) of acrylic acid (distilled) or allyl amine in water.The composites were incubated in these solutions for 3 hours at 30 Cthen washed extensively with water and dried in air. Streptavidin wasimmobilized through grafted and/or polymerized acrylic acid moieties byactivating the carboxylic acid groups as NHS esters by a methodanalogous to that described in Example 6.69. Thiol-labeled streptavidinwas immobilized through grafted and/or polymerized allyl amine moietiesafter introducing maleimide groups on the composite by a methodanalogous to that described in Example 6.70. We showed that thesecomposites were capable of binding biotin-labeled reagents and werecapable of exciting ECL from ECL labels; we measured ECL from thecomposite electrodes after treating the materials with an excess ofrabbit IgG that was labeled with biotin and TAG1 groups. ECL from boundIgG was generated by contacting the composite electrode with a solutioncontaining tripropylamine (Assay Buffer, IGEN) and scanning thepotential at the composite electrode from 0 V to −0.8 V to 2.3 V (vs.Ag/AgCl) at a scan rate of 0.1 V/s. The acrylic acid and allyl aminetreated composites yielded, respectively, integrated PMT currents of 203nAs and 123 nAs.

6.75. Use of Plasma to Bond Avidin to Fibril Composites for ECLImmunoassays

Plasma was used to fuse avidin to the surface of composite material. Thefusion was performed by first soaking a block of fibril-EVA composite(example 6.68) composite in a solution of 0.5 mg/ml avidin for 3 hours.After washing with three changes of 60 ml PBS-1, the composite was airdried, cut into strips and treated with either Ar or O₂ plasma for 5minutes at 600 Watts (3000 Wmin). These experiments were conducted on aSeries B plasma Reactor (APS Technologies).

Following plasma treatment of the composite, 5/16″ disks were punchedout and the amount of active avidin immobilized was measured byradiolabeling experiments that used binding biotinylated ¹²⁵I-IgG.Composites with avidin bonded to their surfaces bound 0.244 pmole (Arplasma treated) and 1.899 pmole (oxygen plasma treated) of IgG.

Binding assays using biotinylated-TAG1-IgG (BTI) were also performed.BTI was used at a concentration of 41 nM and the amount bound wasquantitated by ECL. In the case of O₂ plasma fused avidin, 4 μM d-biotinwas incubated with some of the chips for 1 hour prior to the incubationwith BTI. This was done in order to evaluate the amount of binding thatwas due to the interaction between biotin and avidin. The measured ECL:signals for Ar plasma and O₂ plasma were 10424 nAsec and 8179 nAsec,respectively.

6.76. Use of Plasma To Bond Affinity Matrices to Fibril Composites

Acrylic beads (125 mg) bearing biotin (Sigma#B-3272, lot#57F4034) wereground finely. This powder was suspended in approximately 20 ml ofde-ionized water and mixed well on a vortexer; the suspension wasremoved and filtered though a 0.45 micron filter (Gelman Sciences#4598). The filtered suspensions were dried on several 5/16″-diameterdisks of a fibril-EVA composite (27% fibrils by weight, see example6.68).

Some of the disks were plasma treated with O₂ plasma for 10000 Wmin;others were plasma treated with O₂ for 120000 Wmin. After plasmatreatment, the disks were washed 3 times for 10 min in PBS-1 and thenrinsed 3 times with PBS-1 to remove any unbound fragments.

After washing, the derivatized composites were incubated with 0.2mg/mlstreptavidin for 2 hours at room temperature, rinsed and stored in PBS-1buffer. Disks ( 3/16″ diameter) of streptavidin-coated composites werepunched out and placed in the wells of a 96-well plate. A solutioncontaining biotinylated anti-AFP antibody was added to each well. Thedisks were incubated with shaking for 30 min, then washed with PBS-1buffer. 100 μl of TAG1-labeled anti-AFP antibody and 20 μl of samplescontaining known concentrations of AFPwere added to the wells andincubated for 1 hr at room temperature with shaking. The disks were thenrinsed 3 times with assay buffer and ECL was measured as described inExample 6.71. FIG. 66 shows a plot of the log of the ECL signal as afunction of the concentration of AFP in the samples.

6.77. ECL-Based Binding Assays Using Dried Reagents and Not ReQuiring aWash Step

Fibril-EVA composite (prepared as described in Example 6.68) wasoxidized in an oxygen plasma and coated with streptavidin (as describedin Example 6.69). The composite electrode was cut into 5/16″ diameterdisks that were placed in holders that allowed one surface of the disksto be placed in contact with solutions. The disks were treated with 100uL of a solution containing a biotin-labeled anti-AFP antibody (7.5ug/mL, Boehringer-Mannheim) for 1 hour with agitation and then washedwith phosphate-buffered saline. The other reagents required for theassay were then dried on the surfaces by adding and lyophilizing thefollowing solution: TAG1-labeled anti-AFP antibody (12 ug/mL,Boehringer-Mannheim), phosphate (200 mM), tripropylamine (200 mM),bovine serum albumin (2%), sucrose (2%), chloroacetamide (0.1%), andTriton X-100 (0.02%), pH of 7.6. AFP assays were conducted by adding a95 uL sample to the dried reagents on the surface of the disks andincubating for 1 hour while shaking. ECL was excited by inserting acounter and reference electrode into the solution above the compositeelectrodes and scanning the potential at the counter electrode from 0 Vto −0.8 V to 2.3 V (vs. Ag/AgCl) at a scan rate of 4.8 V/s. FIG. 67shows the ECL signals measured at a photomultiplier tube for solutionscontaining known amounts of AFP.

7. INCORPORATION OF REFERENCES

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the forgoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theclaims. Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

1-108. (canceled)
 109. A method for detecting or measuring an analyte ofinterest in a sample in an electrochemiluminescence binding assaycomprising: (a) adding a liquid sample containing an analyte of interestto an assay cassette that comprises (i) an assay electrode; and (ii) adry reagent composition comprising at least one member of the groupconsisting of (1) a labeled binding reagent comprising anelectrochemiluminescent label and (2) an electrochemiluminescencecoreactant; (b) reconstituting said dry reagent composition in saidliquid sample; (c) contacting said electrode with said reconstituted dryreagent composition; (d) applying a voltage waveform effective totrigger electrochemiluminescence at said electrode; and (e) detecting ormeasuring electrochemiluminescence; wherein said detected or measuredelectrochemiluminescence correlates to the presence or amount of saidanalyte in said sample.
 110. The method of claim 109, wherein said dryreagent composition comprises said labeled binding reagent.
 111. Themethod of claim 1 10, wherein said reconstituted dry reagent compositionis not washed from said electrode prior to step (d).
 112. The method ofclaim 110, wherein said electrode and dry reagent composition arepresent in the same reaction enclosure such that steps (b) and (c) occurconcurrently.
 113. The method of claim 110, wherein said cassette is amulti-well plate.
 114. The method of claim 1 10, wherein said electrodeis a carbon composite electrode and said label is an organometallicruthenium complex.
 115. The method of claim 109, wherein said electrodehas a binding reagent immobilized thereon.
 116. The method of claim 109,wherein said dry reagent composition contains at least one additionalcomponent selected from the group consisting of blocking agents, sugars,polysaccharides, surfactants, preservatives or buffering salts.
 117. Themethod of claim 109, wherein said dry reagent composition comprises saidelectrochemiluminescence coreactant.
 118. The method of claim 115,wherein said reconstituted dry reagent composition is not washed fromsaid electrode prior to step (d).
 119. The method of claim 1 15, whereinsaid electrode and dry reagent composition are present in the samereaction enclosure such that steps (b) and (c) occur concurrently. 120.The method of claim 115, wherein said cassette is a multi-well plate.121. The method of claim 115, wherein said electrochemiluminescencecoreactant is a tertiary amine.
 122. The method of claim 121, whereinsaid amine is tripropylamine.
 123. The method of claim 109, wherein saidelectrode is a carbon composite electrode and saidelectrochemiluminescent label is an organometallic ruthenium complex.124. The method of claim 115, wherein said dry reagent compositioncontains at least one additional component selected from the groupconsisting of blocking agents, sugars, polysaccharides, surfactants,preservatives or buffering salts.
 125. The method of claim 109, whereinsaid dry reagent composition comprises said labeled binding reagent andsaid electrochemiluminescence coreactant.
 126. The method of claim 125,wherein said reconstituted dry reagent composition is not washed fromsaid electrode prior to step (d).
 127. The method of claim 125, whereinsaid electrochemiluminescence coreactant is a tertiary amine.
 128. Themethod of claim 127, wherein said amine is tripropylamine.
 129. Themethod of claim 125, wherein said label is a organometallic rutheniumcomplex and said electrode is a carbon composite electrode.
 130. Themethod of claim 125, wherein said electrode and dry reagent compositionare present in the same reaction enclosure such that steps (b) and (c)occur concurrently.
 131. The method of claim 125, wherein said cassetteis a multi-well plate.
 132. The method of claim 125, wherein saidelectrode has a binding reagent immobilized thereon.
 133. The method ofclaim 125, wherein said dry reagent composition contains at least oneadditional component selected from the group consisting of blockingagents, sugars, polysaccharides, surfactants, preservatives or bufferingsalts.
 134. A cassette for detecting or measuring an analyte of interestin a sample in an electrochemiluminescence binding assay comprising: (a)an assay electrode; and (b) a dry reagent composition comprising atleast one member of the group consisting of i) a labeled binding reagentcomprising an electrochemiluminescent label and ii) anelectrochemiluminescence coreactant.
 135. The cassette of claim 134,wherein said dry reagent comprises said labeled binding reagent and saidelectrochemiluminescence coreactant.
 136. The cassette of claim 135,wherein said label is a organometallic ruthenium complex and saidcoreactant is a tertiary amine.
 137. The cassette of claim 136, whereinsaid electrode has a binding reagent immobilized thereon.
 138. Thecassette of claim 137, wherein said electrode is a carbon compositeelectrode.
 139. The cassette of claim 134, wherein said cassette is amulti-well plate.